Many different mathematical models have been developed to describe the mechanical behavior of connective tissues. The mechanical properties of connective tissues depend on various factors such as the properties of the structural components. Microstructural models are the most promising of the mathematical models since these models relate the tissue response in terms of the tissue’s structural components. However, since the behavior and structure of connective tissues are complex, idealizations about the tissue microstructure and interactions are made to enable development of microstructural models. In our study we reviewed the usefulness of a three-parameter microstructural model to describe the microstructural response of ligaments subjected to different levels of strain and cyclic elongation;​ common physiological loading scenarios that the ligament is subjected to on a regular basis. We found that the microstructural model was not useful in describing the response of ligaments subjected to these common loading scenarios. We also noted that the model was limited in describing tissue response. The model required a well defined toe and linear region to predict the microstructural behavior of tissues;​ we did not obtain useful information about specimens strained <8%, levels which are well below reported failure strain levels. We attribute this limitation to one of the assumptions originally made in the development of this model that the behavior of connective tissues subjected to uniaxial loading can be described solely by the recruitment of collagen fiber crimp. We investigated other possible microstructural changes that may contribute to the overall response observed when ligaments are subjected to elongation and repetitive elongation by studying collagen fiber organization. Our studies investigating fiber organization are the first to measure and rigorously quantify the fiber orientation in connective tissues as a function of strain and cyclic elongation. We determined the functional relationship between fiber orientation and strain, as well as fiber orientation and cyclic elongation. We also determined that age effects this functional relationship. In addition to this, we also studied the changes in the tissue ultrastructure by examining the changes in the molecular assembly (D-period) of collagen subjected to different magnitudes of strain and we concluded that the periodicity o f collagen is directly affected by localized strain experienced in the ligament The testing method we employed for this study investigating the changes in the molecular assembly was more advantageous than previously used methods because we captured in-situ changes in the tissue ultrastructure, unlike other studies. This study also provided insight into the possible mechanisms responsible for injuries and reasons for preferential location of these injuries observed clinically.

The cause for the non-linear response of connective tissues is still not fully understood since the structure-fimction relationship in these tissues is unclear. The studies we conducted have provided insight into the structural changes that contribute to the mechanical response of connective tissues. The changes in the fiber orientation observed as a function of strain and cyclic elongation and the location dependent changes observed in the molecular assembly showed that the structural hierarchy of tissues are indeed complex and that the interactions within the collagen structure still needs to be studied in more detail. Also, the studies undertaken lead us to question the role of other structural components, specifically the ground substance, in terms of over all tissue behavior and therefore studies investigating the interactions between structural components (collagen fibers and ground substance) are necessary to enable further development of models.