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Like the human anterior cruciate ligament (ACL), the porcine ACL also has a double bundle structure and several biomechanical studies using this model have been carried out to show the differential effect of these two bundles on macro-level knee joint function. It is hypothesised that if the different bundles of the porcine ACL are mechanically distinct in function, then a multi-scale anatomical characterisation of their individual enthesis will also reveal significant differences in structure between the bundles. Twenty-two porcine knee joints were cleared of their musculature to expose the intact ACL following which ligament-bone samples were obtained. The samples were fixed in formalin followed by decalcification with formic acid. Thin sections containing the ligament insertion into the tibia were then obtained by cryosectioning and analysed using differential interference contrast (DIC) optical microscopy and scanning electron microscopy (SEM). At the micro-level, the anteromedial (AM) bundle insertion at the tibia displayed a significant deep-rooted interdigitation into bone, while for the posterolateral (PL) bundle the fibre insertions were less distributed and more focal. Three sub-types of enthesis were identified in the ACL and related to (i) bundle type, (ii) positional aspect within the insertion, and (iii) specific bundle function. At the nano-level the fibrils of the AM bundle were significantly larger than those in the PL bundle. The modes by which the AM and PL fibrils merged with the bone matrix fibrils were significantly different. A biomechanical interpretation of the data suggests that the porcine ACL enthesis is a specialized, functionally graded structural continuum, adapted at the microto-nano scales to serve joint function at the macro level.

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A novel indentation method was used to investigate the response of articular cartilage in the non-directly loaded region. The indenter contained a relief channel that allowed a tissue bulge to develop within it under load. Healthy bovine tissue samples were statically loaded at a nominal compressive stress of 3.6 MPa. The tissue's equilibrium deformed state was chemically fixed. Differential interference contrast microscopy was used to obtain highresolution images of the deformed microstructure in the region of the tissue bulge.At the submicro-level, the fibrillar resistance to load is highly complex such that regions of relative compression and tension coincide along a single radial direction. This fibrillar level response is manifested as large-scale matrix shear effects within the bulge region. Further, the surface layer, besides being strain-limiting in the tangential direction, has an intrinsic resistance to axial load. Finally, the pattern of load-induced fluid flow is seen to traverse zonal depths and hence suggests an added complexity to the overall permeability in the deformed tissue matrix.

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