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

This study tested whether intermittent cyclic tensile loading accelerates hierarchical collagen fiber maturation in engineered ligament constructs formed from high-density, cell-laden type I collagen gels seeded with neonatal bovine ACL fibroblasts. Constructs were cultured clamped to guide alignment and compared against constructs subjected to cyclic stretch at either 5% or 10% strain for up to 6 weeks. Cyclic loading increased construct contraction (area), accelerated collagen organization across fibril,fiber,and early fascicle length scales, and promoted crimp formation; 10% strain produced larger fibril/fiber diameters and more pronounced, regularly spaced crimp, with fibril/fiber diameters reaching neonatal ACL ranges by 6 weeks. Mechanical property outcomes were strain- and time-dependent: both 5% and 10% loading improved toe-region properties (toe modulus and transition stress) consistent with functional crimp development, but 5% loading yielded the greatest late-stage improvements in elastic modulus, ultimate tensile strength, and collagen accumulation, matching or exceeding immature ACL benchmarks. Acellular gels subjected to the same loading showed no organizational or mechanical improvements, demonstrating that maturation is cell-driven. Composition and LOX activity trends suggested a shift in mechanotransduction with construct maturation: early loading was more catabolic in unorganized gels, whereas later loading became more anabolic once cells were anchored to aligned fibrils/fibers.