Enthesis maturation in engineered ligaments is differentially driven by loads that mimic slow growth elongation and rapid cyclic muscle movement.

Entheses are complex attachments that translate load between elastic-ligaments and stiff-bone via organizational and compositional gradients. Neither natural healing, repair, nor engineered replacements restore these gradients, contributing to high re-tear rates. Previously, we developed a culture system which guides ligament fibroblasts in high-density collagen gels to develop early postnatal-like entheses, however further maturation is needed. Mechanical cues, including slow growth elongation and cyclic muscle activity, are critical to enthesis development in vivo but these cues have not been widely explored in engineered entheses and their individual contribution to maturation is largely unknown. Our objective here was to investigate how slow stretch, mimicking ACL growth rates, and intermittent cyclic loading, mimicking muscle activity, individually drive enthesis maturation in our system so to shed light on the cues governing enthesis development, while further developing our tissue engineered replacements. Interestingly, we found these loads differentially drive organizational maturation, with slow stretch driving improvements in the interface/enthesis region, and cyclic load improving the ligament region. However, despite differentially affecting organization, both loads produced improvements to interface mechanics and zonal composition. This study provides insight into how mechanical cues differentially affect enthesis development, while producing some of the most organized engineered enthesis to date. STATEMENT OF SIGNIFICANCE: Entheses attach ligaments to bone and are critical to load transfer; however, entheses do not regenerate with repair or replacement, contributing to high re-tear rates. Mechanical cues are critical to enthesis development in vivo but their individual contribution to maturation is largely unknown and they have not been widely explored in engineered replacements. Here, using a novel culture system, we provide new insight into how slow stretch, mimicking ACL growth rates, and intermittent cyclic loading, mimicking muscle activity, differentially affect enthesis maturation in engineered ligament-to-bone tissues, ultimately producing some of the most organized entheses to date. This system is a promising platform to explore cues regulating enthesis formation so to produce functional engineered replacements and better drive regeneration following repair.