Structure-Function Changes in Late Stages of Tendon Development and its Sensitivity to Musculoskeletal ActivityPeterson, Benjamin TendonTendon DevelopmentMultiscale MechanicsBioengineeringPHDTendons are essential connective tissues that transmit forces between muscle and bone, ultimately enabling locomotion. These load-bearing capabilities are initially acquired during embryonic and neonatal stages of development as the tissue undergoes the coordinated synthesis and deposition of collagen. While significant progress has been made over the last three decades to unravel this process, there are still several critical questions that remain unanswered. More specifically, during lates stages of chick development there is a near-spontaneous transformation in the load bearing capabilities of embryonic tendons. Prior work has suggested that this transformation is mediated by changes in collagen fibril lengths in vivo. Despite this, no study has directly investigated how changes in the collagenous ultrastructure facilitate this transformation in mechanical capabilities. Prior work has further suggested that this transformation may be mediated via musculoskeletal activity during development. Indeed, while muscle activity has been correlated with gross changes in tendon mass, it’s still unclear how musculoskeletal stimulation drives this transformation in load-bearing capabilities during development. Addressing these unresolved questions is essential to elucidating the biological mechanisms that govern the development of functional loading-bearings. As such, the primary objective of this dissertation is to investigate the multiscale mechanical microenvironment during standard physiological development, determine the gross role of musculoskeletal activity, and probe the structural elements that enable the functional load-bearing capabilities of tendon during late stages of development. Initial work was conducted in mature rat tail tendons to establish the relationship between fibril continuity and multiscale mechanical behavior. We demonstrated that we could increase interfibrillar loading behavior by artificially inducing fibril continuity, establishing that the multiscale strain behavior can be utilized to indirectly evaluate changes in relative collagen fibril lengths in developmental systems. Utilizing these techniques in chick embryos, we observed that an increase in the macroscale modulus with development is accompanied by than an increase in the interfibrillar loading, consistent with an increase in collagen fibril lengths. Suggesting that collagen fibril fusion (i.e., lengthening) drives the iv transformation in developing tendons. Ensuing studies leveraged paralysis agents to probe the role of muscle activity in mediating this developmental response. After immobilization, there was a decrease in macroscale modulus and interfibrillar loading capabilities, suggesting that the transformation in tendon’s load-bearing capabilities is mediated by mechanobiological activity. However, recent work has suggested that crosslinking may contribute to the maturation in the structure-function behavior during tendon development and be dependent on musculoskeletal activity. Thus, subsequent work leveraged a crosslinking inhibitor to investigate the role of intermolecular crosslinks in tendon development. Interestingly, the absence of crosslinks significantly impaired the macroscale and multiscale loading capabilities of the embryonic tissue, highlighting the importance of crosslinks in tendon development. However, thermodynamic and solubility behavior suggested that immobilized tissue had minimal changes in the relative crosslinking, indicating that paralysis is dominantly impairing fibril fusion (i.e., fibril lengthening) events in vivo. To validate these findings, subsequent work aimed to use serial-block face microscopy to track three-dimensional changes within the collagenous ultrastructure. To overcome preexisting limitations in the field, we leveraged deep learning neural networks for the automatic segmentation of collagen fibrils with over 95% accuracy. Two-dimensional characterization showed a progressive increase in collagen fibril lengths with development, further suggesting that fibril fusion behavior mediates changes in loading capabilities. Current attempts at automated tracking of fibrils in three-dimensional space have proved difficult and on-going work aims to address current pitfalls. While still in-progress, this is one of the few studies that has successfully leveraged deep learning strategies to study changes within the collagenous ultrastructure. Collectively, these data suggest that fibril fusion behavior enables the functional transformation in load-bearing capabilities during late stages of tendon development and is mediated by mechanobiological activity. This information provides valuable insight into the multiscale structure-function relationships of developing tendon and the biological mechanisms required to produce robust load-bearing tendon.Esther Gomez, Major Field MemberClaire Thomas, Outside Unit & Field MemberSpencer Szczesny, Chair & Dissertation AdvisorPaula Murphy, Special MemberGregory Lewis, Major Field MemberDaniel Hayes, Program Head/Chairopen_access2022-11-22T13:49:45Zhttps://etda.libraries.psu.edu/catalog/21810bep15