Multi-Scale Structural and Morphological Study of the Knee Ligaments and Their Entheses

Abstract

While various structural studies of the ligament and enthesis have been carried out in the past, few have systematically examined the ligament and its anchorage mechanisms across macro-to-micro-to-ultra scalar levels. As a result, a complete picture of how the ligament attaches to the bone, across these scalar levels, has yet to be fully constructed. Of particular interest are the unique insertion types that exist, for example direct versus indirect, and the variation in structure and function within a given ligament, an example being the anterior cruciate ligament (ACL) and its double bundle anatomy. This thesis combines several investigations into the medial collateral ligament (MCL) and the ACL, investigating specifically how their structures vary across scales, between insertion types and within the ligaments themselves. Four studies were performed: i) The structure of the bovine and porcine MCL entheses were studied at multiple scalar levels; ii) The double bundle structure of the porcine and human ACL and their tibial enthesis were investigated at multiple scalar levels; iii) The anchorage mechanisms of the porcine ACL tibial enthesis were investigated systematically at multiple scalar levels; iv) Using collagen crimp as an indicator, the ovine ACL fibre recruitment pattern, as a function of its double-bundle morphology, was investigated at varying knee flexion angles without loading. Macro-scale data was firstly collected using digital photography, then cryo-sectioned, torn and ligament pull-out ACL-to-bone samples were examined using bright field light microscopy, differential interference contrast (DIC) microscopy, and scanning electron microscopy (SEM) to obtain micro-to-ultra scalar level information. The primary findings were as follows:- 1. In the MCL the indirect ligament-tibial anchorage included various localised adaptations suggesting that this type of insertion could be further sub-categorized. 2. The direct insertion of the ACL into bone appears to be modified according to the bundle type, and hence joint function, and this was a consistent finding in both the porcine and human ACLs. 3. The overall anchorage of the porcine ACL into the tibia is achieved through a collection of structural features found at multiple scalar levels, which includes simple fanning-out of the ligament footprint area at the macro-level, down to fine-scale extensive fibrillar interconnectivity at the ultra-structural level. These multiple-scalar structural realities add a new level of understanding to the functionally graded transition from soft ligament to hard bone. 4. Variations in crimp type and frequency were found to exist between the AM and PL bundles, and within each bundle type comprising the ACL. This suggests that these two bundles are designed, at micro-scalar levels, to respond to different mechanical demands at the joint level. It is commonly accepted that the ACL includes two bundles, and their biomechanical functions are highly structure-dependent. Consequently, by studying the structure of the ACL, its crimping morphology and its enthesis at multiple scalar levels, new insights into the different bundles’ biomechanical roles were revealed. This may have important clinical implications: For example, the variations at the micro-scale in both the enthesis and ligament between the ACL bundles may explain why anatomical reconstruction of the ACL with double-bundle surgery, which does not consider micro-scale variations, still results in similar outcomes to single bundle reconstruction. The four studies presented in this thesis are intended to provide detailed multi-scale structural information of the ACL, MCL, and their entheses. These findings should prove useful to future ligament researchers and modellers and thus contribute to the development of ligament injury prevention protocols and improved reconstruction strategies.