Stepped Cyclic Strain, that Increases or Decreases as Hierarchical Collagen Fibers Form, Does not Further Improve Maturation in Engineered Ligaments

The primary source of strength in ligaments and tendons are hierarchically organized collagen fibers. These fibers largely do not regenerate after injury, with repair, nor in engineered replacements, limiting treatment options. Previously, we developed a culture system which guides ACL fibroblasts in high-density collagen gels to form native-size hierarchical fibers over 6 weeks, and demonstrated that intermittent cyclic stretch further improves maturation. However, additional maturation is needed for clinical relevance. Interestingly, we found cyclic load affected cells differentially depending on the degree of organization, with 10% cyclic strain driving early improvements in unorganized gels and 5% strain being more beneficial later in culture once cells were on aligned fibers. Here, we explored whether a stepped cyclic load, that increased or decreased in strain magnitude as collagen fibers developed, further improved maturation. We hypothesized that progressively decreasing cyclic strain as organization increases would drive cells to produce more mature hierarchical fibers, resulting in stronger replacements. Controls had intermittent cyclic stretch at 0, 5, 7, or 10% strain throughout culture, while stepped load constructs were cyclically loaded with a strain that increased or decreased by 2-3% every 2 weeks as constructs matured. Contrary to our hypothesis, neither decreasing nor increasing load led to further tissue maturation. We hypothesize stepped cyclic load may disrupt cellular tensional homeostasis, leading to repeated remodeling of collagen and shifted proteoglycan accumulation. This study provides insight into how stepped cyclic loading affects hierarchical fiber formation and maturation, which will help to engineer stronger replacements and better rehabilitation protocols.