Summary

The presence or absence of crimp within the anterior cruciate ligament (ACL) sub-bundle anatomy was correlated with knee flexion angle changes and provided a measure of differential loading within its sub-bundle microstructure.

Summary

Macroscopic grading, histologic grading, morphometry, mineral analysis, and mechanical testing were performed to better understand the changes that occur in the cartilage, calcified cartilage, and subchondral bone in early osteoarthritis.

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.

Intervertebral disc herniation and internal disc disruption are both thought to be primarily mechanically based pathologies. Although several studies have previously disrupted discs in vitro, none have examined the resulting disruptions microscopically.

The technique of nuclear pressurization was used to mechanically disrupt ovine lumbar motion segments. A hollow injection screw was inserted longitudinally through the inferior vertebra of each motion segment, so that the injection screw’s tip was located in the centre of the nucleus. Through this screw, a radio-opaque gel was gradually injected into each segment’s nucleus until failure occurred, marked by a large drop in nuclear pressure, or focal change to the disc’s periphery. Following mechanical testing, the internal failure characteristics of each motion segment were assessed using micro-CT and microscopy. During nuclear pressurization, motion segments were held in one of four postures:

0° flexion,

Group I (0° flexion; n=12): Discs failed at a mean nuclear pressure of 13.2±2.1MPa. In most cases failure occurred in a diffuse manner via sequential circumferential tears within the posterior annulus. Group II (7° flexion; n=17): Discs failed at a mean nuclear pressure of 11.2±2.5MPa. Compared to the Group I discs, 7° flexion led to the creation of radial tears extending through the central posterior disc wall. Two types of radial tear occurred: mid-axial and annular-endplate. Mid-axial radial tears were confined to the annulus. Annular-endplate radial tears incorporated both annular and endplate failure; endplate failure in these tears always occurred adjacent to the mid-annulus at the cartilaginous/vertebral endplate junction. Group III (10° flexion; n=17): Discs failed at a mean nuclear pressure of 9.8±2.6MPa. Compared to the Group II discs, 3° of additional flexion increased the proportion of annular-endplate radial tears. Group IV (7° flexion + 2° axial rotation; n=25): Discs failed at a mean nuclear pressure of 7.9±2.4MPa. Compared to the Group II discs, the addition of 2° axial rotation significantly decreased the nuclear pressure at which discs failed, and reduced the occurrence of mid-axial radial tears.

Postures that reduced the disc wall’s ability to withstand high nuclear pressures were associated with an increase in the proportion of disc failures that incorporated tears of the cartilaginous endplates, specifically at the cartilaginous/vertebral endplate junction adjacent to the mid-annulus. The robustness of this junction appears to be intimately linked to the robustness of the disc wall.

Most researchers have employed conventional histological and related methods to investigate the complex architecture of the IVD. Recognizing the inherent limitations of these methods we have pioneered new microstructural and micromechanical techniques that have greatly enhanced our understanding of the 3-D architecture of the IVD. Using sectioning planes that take full account of the oblique fibre angles in the annular wall, combined with specialized optical imaging techniques that provide high resolution structural images of fully hydrated thick sections we have described new levels of structural complexity that are clearly implicated in the biomechanical function of this highly complex connective tissue organ.

The primary regions of structural interest are the annulus, the annular-endplate junction and the nucleus-end-plate junction. Within the complex multilayered annular wall we have identified a system of collagen-rich bridging structures that both integrate proximate oblique and counter-oblique layers as well as providing long-range radial continuity across many layers. We argue that this system has an important biomechanical role of lashing alternate ‘like’ layers together whilst providing for some freedom of fibre angle change between immediately adjacent layers coursing in counter oblique directions. Thus, under the deformations generated by direct compressive, bulging, flexion and minor rotational forces, the structural integrity of the annulus is maintained.

We have also clarified important features of both annular/endplate and nucleus/endplate structural integration. Our very recent structural studies of the lumbar motion segment suggest that the current models of disc/endplate integration require substantial revision. This presentation will describe new experimental evidence in support of a more appropriate model of structural integration.

In this microanatomical and biomechanical study we investigated OA lesion sites and the adjacent intact tissue in an attempt to uncover clues of a pre-OA tissue state and its progression to OA.

Bovine patellae (n=30) showing various degrees of degeneration, where lesions were located in the distal-lateral quarter, were used for the microanatomical study. Cartilage-on-bone samples were cut to include one with the lesion site and the other with the adjacent intact site. These blocks were formalin fixed. For the mechanical testing tissue samples (n=20) ranging from intact to mildly through to severely degenerate were statically compressed (7MPa) to near-equilibrium using a cylindrical indenter, and then formalin-fixed to capture this deformed state. Following mild decalcification of both sets of tissues, full-depth cartilage-bone cryo-sections incorporating the intact-lesion transition and the deformation profile were obtained and studied in their fully hydrated state using differential interference contrast optical microscopy (DIC).

There were three mechanically-significant microstructural features of the cartilage-bone system that varied with tissue degeneration:

the integrity of the strain limiting surface layer,

Importantly, our mechanical analysis showed how disruption of the cartilage continuum by surface disruption and matrix fibrillar de-structuring, had wider mechanical consequences at the biologically-active osteochondral junction of the adjacent healthy cartilage. The structural changes in the osteochondral junction beneath the still-intact articular cartilage adjacent to the lesion site included the ‘sprouting’ of bone spicules or cones that were morphologically similar to those associated with primary bone formation.

The microanatomical and micromechanical data suggests that there is a mechanobiological link between the altered microstructural response of degenerate cartilage to load and the way in which structural changes develop in the normal adjacent tissue. We propose that while the progression of OA involves first the processes of new bone formation in tissue adjacent to lesion sites, its initiation is due to a disrupted cartilage matrix that alters a regional mechanical environment that includes adjacent healthy tissue.

The detailed anatomy of interconnectivity of intervertebral disc annular fibre layers remains unclear and a structural survey of interlammellar connectivity is required to understand this anatomy and mechanical behavior. The subsequent failure modes of the annulus under hydrostatic loading require definition to understand genesis of annular tears and disc herniation.

Interlamellar Connectivity. We imaged anterior annular sections from ovine lumbar discs. Using differential interference contrast microscopy we were able to reconstruct a three-dimensional image of the interconnecting bridging network between layers. Annular Disruption. The nuclei of ovine lumbar discs were gradually pressurised to failure by injecting a viscous radio-opaque gel via their inferior vertebrae. Investigation of the resulting annular disruption was carried out using micro-computed tomography and DIC microscopy. This allowed analysis of annular failure patterns and herniation, with analysis of the pathway of nuclear movement during prolapse in relation to annular fibre separation within and between fibre layers.

Interlamellar Connectivity. A high level of connectivity between apparently disparate bridging elements was revealed. The extended form of the bridging network is that of occasional substantial radial connections spanning many lamellae with a subsidiary fine branching network. The fibrous bridging network is highly integrated with the lamellar architecture via a collagen-based system of interconnectivity. In particular this bridging network appears to have a major role in anchoring leading edges of incomplete annular lamellae. Annular Disruption and Disc Herniation. Gel extrusion from the posterior annulus was the most common mode of disc failure. Unlike other regions of the annular wall, the posterior region was unable to distribute hydrostatic pressures circumferentially. In each extrusion case, severe disruption to the posterior annulus was observed. While intralamellar disruption occurred in the mid annulus, interlamellar disrupt ion occurred in the outer posterior annulus. Radial ruptures between lamellae always propagated in the mid-axial plane.

The interlamellar architecture of the annulus is far more complex than has previously been recognised and this paper further defines the microanatomy of the disc wall. The hydrostatic pressure failure mode of the posterior annulus mirrors clinic al sites of annular tear and disc prolapsed in the neutral loading position.

Introduction: The basic architecture of the annulus fibrosus has long been established; successive lamellae containing parallel collagen fibers cross obliquely as you move through the annular wall, with the lamellae anchored in the endplates to form a multi-ply structure. Less is known of the interactions between fiber populations in the multi-laminate annulus fibrosus. Their significant contribution to the material behaviour was highlighted in Elliot and Setton’s 2001 attempt to build a material model based on experimental measurements of properties of the annulus. Recent research has confirmed a localized rather than a homogeneous or dispersed mode of interconnectivity between lamellae. Whilst clearly indicating localized bridging structures these studies have allowed only a glimpse of how these bridging elements fit within the overall lamellar architecture. The aim of this investigation was to analyse the interlamellar interconnectivity in its full 3-dimensional form and in complete segments of the annular wall.

Methods: Anterior segments of ovine lumbar discs in two age groups were sectioned along the oblique fiber angle. A 3-dimensional picture of the translamellar bridging network (TLBN) is developed using structural information obtained from fully hydrated unstained serial sections imaged by differential interference contrast optics.

Results: A high level of connectivity between apparently disparate bridging elements was revealed. The extended form of the bridging network is that of occasional substantial radial connections spanning many lamellae with a subsidiary fine branching network. The fibrous bridging network is highly integrated with the lamellar architecture via a collagen-based system of interconnectivity.

Discussion: This study demonstrates a far greater complexity to the interlamellar architecture of the disc annulus than has previously been recognised. Our findings are clearly relevant to disc biomechanics. Significant degrading of the TLBN may result in annular weakening leading potentially to disc failure. Most importantly this work opens the way to a much clearer understanding of the micro-anatomy of the disc wall.

Introduction: Compound mechanical loadings have been used to re-create clinically relevant annular disruptions in vitro. However, the role that individual loading parameters play in disrupting the lumbar disc’s annulus remains unclear. Using the recently described technique of nuclear inflation, the role that elevated nuclear pressures play in disrupting the lumbar intervertebral disc’s annulus fibrosus was investigated.

Methods: The nuclei of 12 ovine lumbar motion segments, posterior elements removed, were gradually pressurized by injecting a viscous radio-opaque gel via an injection screw fitted axially through their inferior vertebrae. Pressurization was conducted until catastrophic failure of the disc occurred. Investigation of the resulting annular disruption was carried out in tandem using micro-computed tomography and differential interference contrast microscopy.

Results: 3 of the 12 motion segments tested were excluded from the results due to improper placement of the injection screw, resulting in pressurization of the inferior vertebra rather than the nucleus. Mean failure pressure of the remaining 9 motion segments was 14.1 ± 3.9 MPa. Peak rates of pressurization ranged from 0.1–0.4MPa/s. Gel extrusion from the posterior annulus occurred in 7 discs and was the most common mode of failure. Unlike other aspects of the annular wall, the posterior region was unable to distribute hydrostatic pressures circumferentially. In each extrusion case, sever disruption to the posterior annulus occurred. While intralamellar disruption occurred in the mid annulus, interlamellar disruption occurred in the outer posterior annulus. Radial ruptures between lamellae always occurred in the mid-axial plane.

Discussion: With respect to the annular wall, the posterior region is most susceptible to failure in the presence of high nuclear pressure, even when loaded in the neutral position. The limited ability of the injected gel to cross the posterior-posterolateral boundaries, effectively concentrating hydrostatic stress within the posterior annulus, indicates that the laminate architecture along these radial lines is of mechanical significance. Within the outer posterior annulus, the prominence of inter-lamellar rather than intralamellar disruption indicates weak interlamellar cohesion. This suggests that nuclear material migrating down a radial fissure may easily track circumferentially within an interlamellar space upon reaching the inner lamellae of the outer annulus. This may explain why the majority of herniations are limited to protrusions contained within the outer annular wall.. The tendency for annular fibres to rupture in the mid-axial plane when loaded hydrostatically suggests that for a radial fissure or herniation to occur at the annular-endplate junction, a compounding bending or torsional load is required.

Introduction Understanding how annular failure might occur following increased nuclear pressurisation requires an experimental approach that avoids artefactual injury to the annulus but reveals structural disruption resulting directly from the pressurisation event. The aim of this study was to investigate the fundamental mechanisms by which both intra and inter-lamellar relationships are disrupted by nuclear pressurisation, with the development of a model that might accurately reproduce mechanisms of intervertebral disc injury secondary to events causing raised intradiscal pressure.

Methods Bovine motion segments were subjected to internal pressurisation using a novel “through vertebra” method. Intra and inter-lamellar sections were deliberately chosen so as to expose systematic patterns of structural disruption resulting from the pressurisation event. This micro-disruption was investigated using a novel method which combined microtensile manipulation and simultaneous differential contrast imaging of the fully hydrated unstained sections.

Results The inner annulus was most severely disrupted. The middle regions developed a series of regular clefts along axes of weakness within the in-plane arrays of fibres in each lamella with a slight oblique passage radially away from the centre. These annular clefts separated the pre-existing transverse or side-to-side interconnections within the longitudinal fibre arrays. Progression to the peripheral lamellae occurred when the clefts crossed lamellae with associated inter-lamellar junction separation, with progressively lesser degrees of disruption further from the central area of pressurisation.

Discussion This study demonstrates that raised intradiscal pressure creates a consistent pattern of annular failure, which may preceed clinically relevant disc lesions, and specifically annular lesions. These findings offer a possible explanation for (a) annular weakening that alters the ability of the nucleus to maintain hydration after load, (b) the initiation of paths for annular tear development, (c) pathways that may expand to allow disc prolapse and (d) pathways for ingrowth of inflammatory and neural tissue mediating disc pain.

Introduction Compressive loads applied to the disc are translated into an internal hydrostatic pressure in the nucleus which is resisted by the annulus. The anisotropic, inhomogeneous, multiply, collagenous architecture of the annulus reflects the complex pattern of mainly tensile stresses developed in this region of the disc during normal function. While many previous investigators have analysed the tensile behaviour of the annulus there still remains much to be learned about the fundamental structural relationships within the disc wall and upon which normal function depends. There is also much to be learned about how alterations in these relationships might lead to disc malfunction. Both intra and inter-lamellar structural relationships will be fundamental to the maintenance of annular wall strength. The aim of this study was to use high resolution ‘live’ imaging to explore the fundamental structural relationships governing the elasticity, intrinsic strength and rupture behaviour of intra-lamellar sections.

Methods In-plane intra-lamellar sections of nominal thickness 70–90μm were cut from the outer lamellae of bovine discs using a sledging microtome. Using a micro-mechanical technique in combination with simultaneous high resolution differential interference contrast optical microscopy (DIC) structural responses both along and transverse to the primary direction of the mono-array of collagen fibres were studied.

Results and Discussion Stretching along the primary alignment direction revealed a biomechanical response consistent with the behaviour of an array of strong fibres whose strength is governed primarily by the strength of embedding in the vertebral endplates rather than from inter-fibre cohesion along their length. The mono-aligned array, even when lacerated, is highly resistant to any further tearing across the alignment direction. Although not visible in the relaxed mono-arrays, transverse stretching revealed a highly complex set of interconnecting structures embodying a series of hierarchical relationships not previously revealed. It is suggested that these structures might play an important role in the containment under pressure of the nuclear contents. The dramatic differences in rupture behaviour observed along versus across the primary fibre direction are consistent with known clinical consequences arising from varying degrees of annular wall damage, and might also explain various types of disc herniation. The lamellar architecture of the healthy disc revealed by this ‘live’ tissue investigation provides an important reference framework for exploring structural changes associated with disc trauma and degeneration.

Introduction The structure of the disc is both complex and inhomogeneous, and it functions as a successful load-bearing organ by virtue of the integration of its various structural regions. These same features also render it impossible to assess the failure strength of the disc from isolated tissue samples which at best can only yield material properties.

Methods This study investigated the intrinsic failure strength of the intact bovine caudal disc under a simple mode of internal hydrostatic pressure. Using a hydraulic actuator, coloured hydrogel was injected under monitored pressure into the nucleus through a hollow screw insert which passed longitudinally through one of the attached vertebrae.

Results Failure did not involve vertebra/endplate structures. Rather, failure of the disc annulus was indicated by the simultaneous manifestation of a sudden loss of gel pressure, a flood of gel coloration appearing in the outer annulus and audible fibrous tearing. A mean hydrostatic failure pressure of 18±3 MPa was observed which was approximated as a thick-wall hoop stress of 45±7 MPa.

Discussion The experiment provides a measurement of the intrinsic strength of the disc using a method of internal hydrostatic loading which avoids any disruption of the complex architecture of the annular wall. Although the disc is subjected to a much more complex pattern of loading than is achieved using simple hydrostatic pressurization, this mode provides a useful tool for investigating alterations in intrinsic disc strength associated with prior loading history or degeneration.