In the treatment of ligament injuries there has been much interest in the restoration of the actual ligament anatomy, and the extent to which the original enthesis may be re-established. This study therefore seeks to uncover new information on ligament microstructure and its insertion into bone.

Five bovine medial collateral ligaments (MCL) and five ovine anterior cruciate ligaments (ACL) were used in this study. All ligaments were harvested with the femoral and tibial bony insertions still intact. The bone ends were clamped and the MCL stretched to about 10% strain while the ACL underwent a 90° twist. The entire ligament-bone system, under load, was fixed in 10% formalin solution for 12 hours, following which it was partially decalcified to facilitate microsectioning. Thin 30 ìm-thick sections of the ligament-bone interface and ligament midsubstance were obtained. Differential Interference Contrast (DIC) optical microscopy was used to image the ligament and bone microarchitecture in the prescribed states of strain.

Fibre crimp patterns were examined for the prescribed loading condition and showed distinct sections of fibre recruitment. Transverse micro-imaging of the ligament showed a significant variation in the sub-bundle cross-sectional area, ranging from 100ìm to 800 ìm. Those bundles closer to the central long axis of the ligament were numerous and small, while moving towards the periphery, they were large and singular. Both classifications of entheses, direct and indirect, were observed in the MCL insertions into the femur and tibia respectively. Of interest was the indirect insertion where the macro-level view of the near parallel attachment of fibres to bone via the periosteum was revealed, at the microscale, to involve a gradually increasing orthogonal insertion of fibres. This unique transition occurred closer to the joint line. In the ACL the anterior-medial (AM) and posterior-lateral (PL) bundles were easily discernable. All insertions into bone for the ACL were of the direct type. Fibres were thus seen to transition through the four zones of gradual mineralization to bone. However the manner in which the AM and PL bundles insert into bone, and the lateral soft tissue transition between these two bundles, revealed a structural complexity that we believe is biomechanically significant.

This ‘mechano-structural’ investigation, using novel imaging techniques, has provided new insights into the microstructure of the ligament bone system. The images presented from this study are aimed to aid new approaches for reconstruction, and provide a blue-print for the design of ligament-bone systems via tissue engineering.

The spinal motion segment relies critically on there being a mechanically robust integration between the compliant disc tissues and the rigid vertebral bone. Achieving such integration represents a major structural challenge. This study explores in detail the microstructural mechanisms involved in both the nucleus-endplate and annulus-endplate regions.

Vertebra-nucleus-vertebra samples were obtained from mature ovine lumbar motion segments and subjected to a novel ring-severing technique designed to eliminate the strain-limiting influence of any remaining annular elements. These samples were loaded in tension and then chemically fixed in order to preserve the stretched fibre arrangement, and then decalcified. Annulus-vertebra samples were similarly treated but without any loading prior to fixation. Differential interference contrast optical microscopy was then used to image at high resolution cryosectioned slices of the still integrated disc-vertebral endplate regions while maintained in their fully hydrated state.

Structural continuity across the nucleus-endplate junction was sufficient for the samples to support, on average, 20 N before tensile failure occurred. Microscopic examination revealed fibres inserting into the endplates and extending continuously from vertebra to vertebra in the central nuclear region. While the fibres in the nucleus possess a significant level of structural integration with the endplates their role is not primarily a tensile one: rather, in combination with their convoluted geometry, they confer on the nucleus a form of ‘tethered’ mobility. This permits a high degree of shape change in the nucleus during normal disc function in which hydrostatic loading plays an essential role. The annular fibre bundles on entering the endplate are shown to subdivide into sub-bundles to form a 3-D multi-leaf morphology with each leaf separated by cartilaginous endplate matrix. This branched morphology increases the interface area between bundle and matrix in proportion to the number of sub-bundles formed.

Our study challenges previously published views on nucleus-endplate relationships. We also show that the robust integration of the annular fibres in the endplate is achieved via a branched morphology exploiting a mechanism of shear-stress transfer, with the anchorage strength optimised over a relatively short endplate insertion depth.