A multi-scale structural study of the porcine anterior cruciate ligament tibial enthesis

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 micro-to-nano scales to serve joint function at the macro level.