Protein Levels and Microstructural Changes in Localized Regions of Early Cartilage Degeneration Compared with Adjacent Intact Cartilage.

OBJECTIVE: It was hypothesized that the respective protein profiles of bovine cartilage from sites of localized mild to moderate (GI to GII) degeneration versus adjacent sites of intact tissue would vary in accordance with the tissue microstructural changes associated with a pre-osteoarthritic state. METHODS: A total of 15 bovine patellae were obtained for this study. Paired samples of tissue were collected from the lateral region of each patella. If the patella contained a site of degeneration, a paired tissue set involved taking one sample each from the degenerated site and the intact tissue adjacent to it. Sufficient tissue was collected to facilitate 2 arms of investigation: microstructural imaging and proteome analysis. The microstructural analysis used a bespoke tissue preparation technique imaged with differential interference contrast optical microscopy to assess fibrillar scale destructuring and underlying bone spicule formation. An iTRAQ-based proteome analysis was performed using liquid chromatography-tandem mass spectrometry to identify the differential levels of proteins across the intact and degenerated cartilage and further, the results were validated with multiple reaction monitoring assay. RESULTS: In the healthy cartilage pairs, there was no significant variation in protein profiles between 2 adjacent sample sites. In pairs of tissue that contained a sample of GI/GII tissue, there were both significant microstructural changes as well as the difference in abundance levels of 24 proteins. CONCLUSIONS: From the known functions of the 24 proteins, found to be strongly aligned with the specific microstructural changes observed, a unique "proteins ensemble" involved in the initiation and progression of early cartilage degeneration is proposed.

Sagittal Alignment With Downward Slope of the Lower Lumbar Motion Segment Influences Its Modes of Failure in Direct Compression: A Mechanical and Microstructural Investigation.

STUDY DESIGN: Microstructural investigation of compression-induced herniation of ovine lumbar discs with and without added component of anterior-inferior slope. OBJECTIVE: Does increased shear arising from a simulated component of motion segment slope imitating sacral slope weaken the lateral annulus and increase risk of overt herniation at this same region. SUMMARY OF BACKGROUND DATA: An increase in sacral slope secondary to lordosis and pelvic incidence increases shear stresses at the lumbosacral junction and has been associated with an increase in spondylolisthetic disorders and back injury. The small component of forward shear induced when a segment is compressed in flexion is suggested to cause differential recruitment of the lateral annular fibers leading to its early disruption followed by intra-annular nuclear tracking to the posterolateral/posterior regions. However, the influence of even greater forward shear arising from the added component of slope seen where pelvic incidence and lumbar lordosis are increased in the lower lumbar spine is less understood. METHODS: Ovine motion segments were compressed at 40 mm/min up to failure; 9 with a horizontal disc alignment and 26 with a segment slope of 15° and then analyzed structurally. RESULTS: All the horizontal discs failed (11.8 ±â€Š2.4 kN) via vertebral fracture without any evidence of soft tissue failure even in the lateral aspects of the discs. The increased forward shear resulting from the slope decreased the failure load (6.4 ±â€Š1.6 kN). The sloping discs mostly suffered mid-span, noncontinuous disruption of the lateral annulus with some extruding nuclear material directly from these same lateral regions. CONCLUSION: The increased level of forward shear generated in moderately sloping lumbar segments when compressed was abnormally damaging to the lateral regions of the disc annulus. This is consistent with the view that shear differentially loads the oblique-counter oblique fiber sets in the lateral annulus, increasing its vulnerability to early disruption and overt herniation. LEVEL OF EVIDENCE: N/A.

New evidence for structural integration across the cartilage-vertebral endplate junction and its relation to herniation.

BACKGROUND CONTEXT: The cartilaginous and bony material that can be present in herniated tissue suggests that failure can involve both cartilaginous and vertebral-endplates. How structural integration is achieved across the junction between these two distinct tissue regions via its fibril and mineral components is clearly relevant to the modes of endplate failure that occur. PURPOSE: To understand how structural integration is achieved across the cartilaginous-vertebral endplate junction. STUDY DESIGN: A micro- and fibril-level structural analysis of the cartilage-vertebral endplate region was carried out using healthy, mature ovine motion segments. METHODS: Oblique vertebra-annulus-vertebra samples were prepared such that alternate layers of lamellar fibers extended from vertebra to vertebra. The endplate region of each sample was then decalcified in a targeted manner before being loaded in tension along the fiber direction to achieve incomplete rupture within the region of the endplate. The failure regions were then analyzed with differential interference contrast microscopy and scanning electron microscopy. RESULTS: Microstructural analysis revealed that failure within the endplate region was not confined to the cement line. Instead, rupture continued into the underlying vertebral endplate with bony material still attached to the now unanchored annular bundles. Ultrastructural analysis of the partially ruptured regions of the cement line revealed clear evidence of blending/interweaving relationships between the fibrils of the annular bundles, the calcified cartilage and the bone with no one pattern of association appearing dominant. These findings suggest that fibril-based structural cohesion exists across the cement line at the site of annular insertion, with strengthening via a mechanism somewhat analogous to steel-reinforced concrete. The fibrils are brought into a close intermingling association with interfibril forces mediated via the mineral component. CONCLUSIONS: This study provides clear evidence of structural connectivity across the cartilaginous-vertebral endplate junction by the intermingling of their fibrillar components and mediated by the mineral phase. This is consistent with the clinical observation that in some disc herniations bony material can be still attached to the extruded soft tissue.

The Influence of Concordant Complex Posture and Loading Rate on Motion Segment Failure: A Mechanical and Microstructural Investigation.

STUDY DESIGN: Microstructural investigation of compression-induced herniation of a lumbar disc held in a concordant complex posture. OBJECTIVE: To explore the significance of loading rate in a highly asymmetric concordant posture, comparing the mechanisms of failure to an earlier study using a nonconcordant complex posture. SUMMARY OF BACKGROUND DATA: A recent study with a nonconcordant complex posture (turning in the opposite direction to that which the load is applied) demonstrated the vulnerability of the disc to loading that is borne by one set of oblique-counter oblique fiber sets in the alternating lamellae of the annulus, and aggravated by an elevated loading rate. Given the strain rate-dependent properties of the disc it might be expected that the outcome differs if the posture is reversed. METHODS: Forty-one motion segments from ovine 16 spines were split into two cohorts; adopting the previously employed low rate (40 mm/min) and surprise rate (400 mm/min) of loading. Both groups of damaged discs were then analyzed microstructurally. RESULTS: With the lower rate loading the concordant posture significantly reduced the load required to cause disc failure than earlier described for nonconcordant posture (6.9 vs. 8.4 kN), with more direct tears and alternate lamella damage extending to the anterior disc. Contrary to this result, with a surprise rate, the load at failure was significantly increased with the concordant posture (8.08 vs. 6.96 kN), although remaining significantly less than that from a simple flexed posture (9.6 kN). Analysis of the damage modes and postures suggest facet engagement plays a significant role. CONCLUSION: This study confirms that adding shear to the posture lowers the load at failure, and causes alternate lamella rupture. Load at failure in a complex posture is not determined by loading rate alone. Rather, the strain rate-dependent properties of the disc influence which elements of the system are brought into play. LEVEL OF EVIDENCE: N/A.

Does an Annular Puncture Influence the Herniation Path?: An In Vitro Mechanical and Structural Investigation.

STUDY DESIGN: A study of mechanically induced herniation in punctured ovine discs followed by structural analysis. OBJECTIVE: To investigate whether an annular puncture influences the path that herniation takes by providing direct passage for nucleus through the annulus and therefore whether it increases the risk of acute herniation from overload at the site of damage independent of any longer-term degeneration. SUMMARY OF BACKGROUND DATA: Ten years after treatment with discography both degenerative changes and frequency of herniation have been shown to increase compared to untreated discs. Although the effect of an annular puncture over time has been widely investigated the question of whether it increases the risk of acute herniation has not been resolved. METHODS: The posterolateral annuli of healthy ovine lumbar discs were punctured with either a 25-gauge (n = 8) or a larger 18-gauge (n = 8) needle and then compressed in a flexed posture of 10° until initial indications of failure. The entire volume of the disc was visually assessed for structural damage by obtaining progressive, full transverse cross-sections of its entire height thus exposing all regions of the disc. RESULTS: There was no association between the 25-gauge puncture and disc disruption and herniation. In contrast, nuclear material was observed to migrate through the 18-gauge needle puncture. Disruption of the lateral inner annulus was observed in 12 out of the 16 discs tested. CONCLUSION: The risk of acute herniation through the puncture site is dependent on the needle diameter used. Under the conditions employed the lateral inner annulus remains the site most vulnerable to disruption independent of the presence of a posterolateral puncture. LEVEL OF EVIDENCE: N /A.

A Microstructural Investigation of Disc Disruption Induced by Low Frequency Cyclic Loading.

STUDY DESIGN: Microstructural investigation of low frequency cyclic loading and flexing of the lumbar disc. OBJECTIVE: To explore micro-level structural damage in motion segments subjected to low frequency repetitive loading and flexing at sub-acute loads. SUMMARY OF BACKGROUND DATA: Cumulative exposure to mechanical load has been implicated in low back pain and injury. The mechanical pathways by which cyclic loading physically affects spine tissues remain unclear, in part due to the absence of high quality microstructural evidence. METHODS: The study utilized seven intact ovine lumbar spines and from each spine one motion segment was used as a control, two others were cyclically loaded. Ten motion segments were subjected to 5000 cycles at 0.5 Hz with a peak load corresponding to ∼30% of that required to achieve failure. An additional small group of segments subjected to 10,000 or 30,000 cycles was similarly analyzed. Following chemical fixation and decalcification samples were cryosectioned along one of the oblique fiber angles and imaged in their fully hydrated state using differential interference contrast optical microscopy. Structural damage obtained from the images was organized into an algebraic shell for analysis. RESULTS: At 5000 cycles the disc damage was limited to inner wall distortions, evidence of stress concentrations at bridging-lamellae attachments, and small delaminations. The high-cycle discs tested exhibited significant mid-wall damage. There was no evidence of nuclear material being displaced. CONCLUSION: At this low frequency and without the application of sustained loading or a more severe loading regime, or maintaining a constant flexion with repetitive loading, it seems unlikely that actual nuclear migration occurs. It is possible that the inner-annular damage shown in the low dose group could disrupt pathways for nutrient diffusion leading to earlier cell death and matrix degradation, thus contributing to a cascade of degeneration. LEVEL OF EVIDENCE: N/A.

A more realistic disc herniation model incorporating compression, flexion and facet-constrained shear: a mechanical and microstructural analysis. Part I: Low rate loading.

PURPOSE: To date, the mechanisms of disc failure have been explored at a microstructural level in relatively simple postures. However, in vivo the disc is known to be subjected to complex loading in compression, bending and shear, and the influence of these factors on the mechanisms of disc failure is yet to be described at a microstructural level. The purpose of this study was to provide a microstructural analysis of the mechanisms of failure in healthy discs subjected to compression while held in a complex posture incorporating physiological amounts of flexion and facet-constrained shear. METHODS: 30 motion segments from 10 healthy mature ovine lumbar spines were compressed in a complex posture intended to simulate the situation arising when bending and twisting while lifting a heavy object, and at a displacement rate of 40 mm/min. Nine of the 30 samples reached the predetermined displacement prior to a reduction in load and were classified as early-stage failures, providing insight into initial areas of disc disruption. Both groups of damaged discs were then analysed microstructurally using light microscopy. RESULTS: Complex postures significantly reduced the load required to cause disc failure than earlier described for flexed postures [8.42 kN (STD 1.22 kN) compared to 9.69 kN (STD 2.56 kN)] and resulted in a very different failure morphology to that observed in either simple flexion or direct compression, involving infiltration of nucleus material in a circuitous path to the annular periphery. CONCLUSION: The complex posture as used in this study significantly reduced the load required to cause disc failure, providing further evidence that asymmetric postures while lifting should be avoided if possible.

A more realistic disc herniation model incorporating compression, flexion and facet-constrained shear: a mechanical and microstructural analysis. Part II: high rate or 'surprise' loading.

PURPOSE: Part I of this study explored mechanisms of disc failure in a complex posture incorporating physiological amounts of flexion and shear at a loading rate considerably lower than likely to occur in a typical in vivo manual handling situation. Given the strain-rate-dependent mechanical properties of the heavily hydrated disc, loading rate will likely influence the mechanisms of disc failure. Part II investigates the mechanisms of failure in healthy discs subjected to surprise-rate compression while held in the same complex posture. METHODS: 37 motion segments from 13 healthy mature ovine lumbar spines were compressed in a complex posture intended to simulate the situation arising when bending and twisting while lifting a heavy object at a displacement rate of 400 mm/min. Seven of the 37 samples reached the predetermined displacement prior to a reduction in load and were classified as early stage failures, providing insight to initial areas of disc disruption. Both groups of damaged discs were then analysed microstructurally using light microscopy. RESULTS: The average failure load under high rate complex loading was 6.96 kN (STD 1.48 kN), significantly lower statistically than for low rate complex loading [8.42 kN (STD 1.22 kN)]. Also, unlike simple flexion or low rate complex loading, direct radial ruptures and non-continuous mid-wall tearing in the posterior and posterolateral regions were commonly accompanied by disruption extending to the lateral and anterior disc. CONCLUSION: This study has again shown that multiple modes of damage are common when compressing a segment in a complex posture, and the load bearing ability, already less than in a neutral or flexed posture, is further compromised with high rate complex loading.

How a decreased fibrillar interconnectivity influences stiffness and swelling properties during early cartilage degeneration.

OBJECTIVE: The functional coupling between the fibrillar network and the high-swelling proteoglycans largely determines the mechanical properties of the articular cartilage matrix. The objective of this new study was to show specifically how changes in fibrillar interconnectivity arising from early cartilage degeneration influence transverse stiffness and swelling properties at the tissue level. DESIGN: Radial zone transverse layers of cartilage matrix were obtained from intact and mildly degenerate bovine patellae. Each layer was then subdivided to assess tensile stiffness, free-swelling response, glycosaminoglycan (GAG) content, and micro- and ultra-structural features. RESULTS: The tensile modulus was significantly lower and the degree of swelling significantly higher for the degenerate matrix compared to the intact. Scanning electron microscopy revealed a homogeneous response to transverse strain in the intact cartilage, whereas large non-fibrillar spaces between fibril aggregates were visible in the degenerate matrix. Although there were no significant differences in GAG content it did correlate significantly with stiffness and swelling in the intact samples but not in the degenerate. CONCLUSIONS: The lower degree of fibril network interconnectivity in the degenerate matrix led to both a decreased transverse stiffness and reduced resistance to osmotic swelling. This network 'de-structuring' also resulted in a reduced functional interaction between the fibrillar network and the proteoglycans. The study provides new insights into the role of the fibrillar network and how changes in the network arising from the degenerative cascade will influence tissue level behaviour.

Posterolateral Disc Prolapse in Flexion Initiated by Lateral Inner Annular Failure: An Investigation of the Herniation Pathway.

STUDY DESIGN: Structural investigation of mechanically induced herniations in ovine lumbar motion segments. OBJECTIVE: This new study addresses the question of whether there are regions other than the posterior and posterolateral aspects that are implicated in the initiation of disc disruption and herniation. SUMMARY OF BACKGROUND DATA: Flexion in combination with compressive loading will induce disc herniations in healthy motion segments in vitro. Although it is widely accepted that the posterior and posterolateral regions of the disc are the primary sites of herniation much less is known as to whether other regions of the disc might be involved in the herniation process. METHODS: Healthy ovine lumbar motion segments (n = 14) were flexed 10° and compressed at a rate of 40 mm/min up to point of failure. The discs were macroscopically analyzed using progressive transverse sectioning to obtain a more global picture of internal disc disruption and herniation. RESULTS: A high prevalence of disruption in the lateral annulus was found associated with circumferential tracking of nucleus between the annular layers toward the posterolateral and posterior regions. In all tests this lateral disruption did not cause any discernible external change in the lateral disc periphery after the removal of load. After imposing the predetermined flexion the applied compression also induced a forward anterior shear of the superior vertebra of approximately equal magnitude to the axial compressive displacement. CONCLUSION: The vulnerability of the lateral annulus to disruption is thought to arise from the overloading of its differentially recruited oblique/counteroblique fiber sets, this in turn generated by anterior shear developed in the flexed, compressed motion segment. This lateral annular disruption, followed by circumferential tracking of nuclear material and resulting in either contained or uncontained extrusions in the posterior or posterolateral annulus, highlights the complexity of the herniation process. LEVEL OF EVIDENCE: N/A.

Microstructure Variations in the Soft-Hard Tissue Junction of the Human Anterior Cruciate Ligament.

The role of the sub-bundles in the anterior cruciate ligament (ACL) has been defined, such that the anterior-medial bundle directly resists anterior tibial translation while the posterior lateral bundle is involved in rotational stability. With regards to this biomechanical function, much of the previous work on bundle-specific morphology has been carried out on the macroscale, with much less attention given to the micro-to-ultrastructural scalar levels. This is especially true of the enthesis and its microstructure, a biomechanically significant region that has been largely neglected in the published literature dealing with ACL sub-bundle anatomy. In this study, the human ACL tibial enthesis was investigated at multiple scalar levels using differential interference contrast and scanning electron microscopies with the aim of determining whether the sub-bundle ligament structure, and its known macroscale function, is consistent with its micro-architecture at the ligament-bone junction. The investigation found that different ligament insertion morphologies exist between the two bundles, where the AM bundle has more intense interdigitation with the bone matrix than that of the PL bundle. The results suggest that such structure-function relationships, especially across scalar-levels, provide new insight into the significance of the sub-bundle anatomy of the ACL. Anat Rec, 300:1547-1559, 2017. © 2017 Wiley Periodicals, Inc.

The influence of early degenerative changes on the vulnerability of articular cartilage to impact-induced injury.

BACKGROUND: Recently, the structural changes in a bovine model of early degeneration were validated by our research group to be analogous to that in early human osteoarthritis. The hypothesis of this study was that the structural changes associated with increasing levels of degeneration would lead to higher levels of tissue damage in response to impact induced injury. METHODS: A total of forty bovine patellae were obtained for this study. Cartilage-on-bone samples were extracted from the distal lateral quarter, a region known to be affected by varying levels of degeneration. A single impact drop test was applied to these samples delivering 2.3J of energy. A dynamic load cell and image capture at 2000fps allowed for the calculation of the reaction stress and coefficient of restitution. The extent of tissue damage was examined from the micro to ultrastructural levels using differential interference contrast optical microscopy and scanning electron microscopy respectively. FINDINGS: The impact mechanical properties of mildly degenerate articular cartilage were not significantly different but showed a significantly larger amount of structural damage. From comparing the mechanical and structural response of intact and mildly degenerate cartilage, to tissue showing increased macro-scale tissue degeneration, the significance of the surface layer and fibrillar scale transverse interconnectivity in effectively attenuating impact loads is demonstrated in this study. INTERPRETATION: This study shows that even though articular cartilage can appear visibly normal under macroscopic observation, the micro-scale structural changes associated with very early stage osteoarthritis can have a significant effect on its vulnerability to impact damage.

How maturity influences annulus-endplate integration in the ovine intervertebral disc: a micro- and ultra-structural study.

The annulus-endplate anchorage system plays a vital role in structurally linking the compliant disc to its adjacent much more rigid vertebrae. Past literature has identified the endplate as a region of weakness, not just in the mature spine but also in the immature spine. The aim of this structural study was to investigate in detail the morphological changes associated with annulus-endplate integration through different stages of maturity. Ovine lumbar motion segments were collected from two immature age groups: (i) newborn and (ii) spring lamb (roughly 3 months old); these were compared with a third group of previously analysed mature ewe samples (3-5 years). Sections from the posterior region of each motion segment were obtained for microstructural analysis and imaged in their fully hydrated state via differential interference contrast (DIC) optical microscopy. Selected slices were further prepared and imaged via scanning electron microscopy (SEM) to analyse fibril-level modes of integration. Despite significant changes in endplate morphology, the annular fibre bundles in all three age groups displayed a similar branching mechanism, with the main bundle splitting into several sub-bundles on entering the cartilaginous endplate. This morphology, previously described in the mature ovine disc, is thought to strengthen significantly annulus-endplate integration. Its prevalence from an age as young as birth emphasizes the critical role that it plays in the anchorage system. The structure of the branched sub-bundles and their integration with the surrounding matrix were found to vary with age due to changes in the cartilaginous and vertebral components of the endplate. Microscopically, the sub-bundles in both immature age groups appeared to fade into the surrounding tissue due to their fibril-level integration with the cartilaginous endplate tissue, this mechanism being particularly complex in the spring lamb disc. However, in the fully mature disc, the sub-bundles remained as separate entities throughout the full depth of their anchorage into the cartilaginous endplate. Cell morphology was also found to vary with maturity within the cartilaginous matrix and it is proposed that this relates to endplate development and ossification.

Staying connected: structural integration at the intervertebral disc-vertebra interface of human lumbar spines.

PURPOSE: To investigate the microscopic fibrous integration between the intervertebral disc, cartilage endplates and vertebral endplates in human lumbar spines of varying degrees of degeneration using differential interference contrast (DIC) optics. Weakness at these junctions is considered to be an important factor in the aetiology of disc herniations. METHODS: Magnetic resonance images (MRIs) of cadaveric lumbar spines were graded for degeneration and motion segments from a range of degenerative grades isolated and bisected sagittally. Following fixation and decalcification, these were cut into segments containing anterior or posterior annulus fibrosus or nucleus pulposus. The segments were cryo-sectioned and sections visualised using both standard light and DIC microscopy. RESULTS: Detachment at the interface between the disc and vertebrae increased with greater degenerative grade (from 1.9 % in Grade I to 28 % in Grade V), especially at the boundary between the cartilage and vertebral endplates. DIC microscopy revealed the fibrous organisation at the IVD-cartilage endplate interface with structural features, such as annular lamellae branching and nodal insertions in the nucleus pulposus region; these have been previously observed in ovine spines, but were less uniform in humans. Structural integrity of the IVD and cartilage endplate was also lost with increasing degeneration. CONCLUSIONS: This preliminary study shows that microscopic structural features may act to maintain attachment between the IVD and CEP in the human spine. Loss of structural integrity in this region may destabilise the spine, possibly altering the mechanical environment of the cells in the disc and so potentially contribute to the aetiopathogenesis of IVD degeneration.

ISSLS Prize Winner: Vibration Really Does Disrupt the Disc: A Microanatomical Investigation.

STUDY DESIGN: Microstructural investigation of vibration-induced disruption of the flexed lumbar disc. OBJECTIVE: The aim of the study was to explore micro-level structural damage in motion segments subjected to vibration at subcritical peak loads. SUMMARY OF BACKGROUND DATA: Epidemiological evidence suggests that cumulative whole body vibration may damage the disc and thus play an important role in low back pain. In vitro investigations have produced herniations via cyclic loading (and cyclic with added vibrations as an exacerbating exposure), but offered only limited microstructural analysis. METHODS: Twenty-nine healthy mature ovine lumbar motion segments flexed 7° and subjected to vibration loading (1300 ±â€Š500 N) in a sinusoidal waveform at 5 Hz to simulate moderately severe physiologic exposure. Discs were tested either in the range of 20,000 to 48,000 cycles (medium dose) or 70,000 to 120,000 cycles (high dose). Damaged discs were analyzed microstructurally. RESULTS: There was no large drop in displacement over the duration of both vibration doses indicating an absence of catastrophic failure in all tests. The tested discs experienced internal damage that included delamination and disruption to the inner and mid-annular layers as well as diffuse tracking of nucleus material, and involved both the posterior and anterior regions. Less frequent tearing between the inner disc and endplate was also observed. Annular distortions also progressed into a more severe form of damage, which included intralamellar tearing and buckling and obvious strain distortion around the bridging elements within the annular wall. CONCLUSION: Vibration loading causes delamination and disruption of the inner and mid-annular layers and limited diffuse tracking of nucleus material. These subtle levels of disruption could play a significant role in initiating the degenerative cascade via micro-level disruption leading to cell death and altered nutrient pathways. LEVEL OF EVIDENCE: 5.

How a radial focal incision influences the internal shear distribution in articular cartilage with respect to its zonally differentiated microanatomy.

Articular surface fibrillation and the loss of both transverse interconnectivity and zonal differentiation are indicators of articular cartilage (AC) degeneration. However, exactly how these structural features affect the load-redistributing properties of cartilage is still poorly understood. This study investigated how a single radial incision made to varying depths with respect to the primary zones of AC influenced its deformation response to compression. Three depths of incision were applied to cartilage-on-bone tissue blocks: one not exceeding the transition zone; one into the mid-radial zone; and one down to the calcified cartilage. Also included were non-incised controls. All samples were compressed to a near-equilibrium strain using a flat-faced indenter that incorporated a central relief channel within which the incision could be positioned lengthwise along the channel axis. Employing fixation under load followed by decalcification, the structural responses of the cartilage-on-bone samples were investigated. The study provides an analysis of the micro-morphological response that is characteristic of a completely normal cartilage-on-bone system but which contains a defined degree of disruption induced by the focal radial incision. The resulting loss of transverse continuity of the cartilage with respect to its zonally differentiated structure is shown to lead to an altered pattern of internal matrix shear whose intensity varies with incision depth.

Derivation of inter-lamellar behaviour of the intervertebral disc annulus.

The inter-lamellar connectivity of the annulus fibrosus in the intervertebral disc has been shown to affect the prediction of the overall disc behaviour in computational models. Using a combined experimental and computational approach, the inter-lamellar mechanical behaviour of the disc annulus was investigated under conditions of radial loading. Twenty-seven specimens of anterior annulus fibrosus were dissected from 12 discs taken from four frozen ovine thoracolumbar spines. Specimens were grouped depending on their radial provenance within the annulus fibrosus. Standard tensile tests were performed. In addition, micro-tensile tests under microscopy were used to observe the displacement of the lamellae and inter-lamellar connections. Finite elements models matching the experimental protocols were developed with specimen-specific geometries and boundary conditions assuming a known lamellar behaviour. An optimisation process was used to derive the interface stiffness values for each group. The assumption of a linear cohesive interface was used to model the behaviour of the inter-lamellar connectivity. The interface stiffness values derived from the optimisation process were consistently higher than the corresponding lamellar values. The interface stiffness values of the outer annulus were from 43% to 75% higher than those of the inner annulus. Tangential stiffness values for the interface were from 6% to 39% higher than normal stiffness values within each group and similar to values reported by other investigators. These results reflect the intricate fibrous nature of the inter-lamellar connectivity and provide values for the representation of the inter-lamellar behaviour at a continuum level.

ISSLS Prize Winner: A Detailed Examination of the Elastic Network Leads to a New Understanding of Annulus Fibrosus Organization.

STUDY DESIGN: Investigation of the elastic network in disc annulus and its function. OBJECTIVE: To investigate the involvement of the elastic network in the structural interconnectivity of the annulus and to examine its possible mechanical role. SUMMARY OF BACKGROUND DATA: The lamellae of the disc are now known to consist of bundles of collagen fibers organized into compartments. There is strong interconnectivity between adjacent compartments and between adjacent lamellae, possibly aided by a translamellar bridging network, containing blood vessels. An elastic network exists across the disc annulus and is particularly dense between the lamellae, and forms crossing bridges within the lamellae. METHODS: Blocks of annulus taken from bovine caudal discs were studied in either their unloaded or radially stretched state then fixed and sectioned, and their structure analyzed optically using immunohistology. RESULTS: An elastic network enclosed the collagen compartments, connecting the compartments with each other and with the elastic network of adjacent lamellae, formed an integrated network across the annulus, linking it together. Stretching experiments demonstrated the mechanical interconnectivities of the elastic fibers and the collagen compartments. CONCLUSION: The annulus can be viewed as a modular structure organized into compartments of collagen bundles enclosed by an elastic sheath. The elastic network of these sheaths is interconnected mechanically across the entire annulus. This organization is also seen in the modular structure of tendon and muscle. The results provide a new understanding annulus structure and its interconnectivity, and contribute to fundamental structural information relevant to disc tissue engineering and mechanical modeling. LEVEL OF EVIDENCE: N/A.

"Surprise" Loading in Flexion Increases the Risk of Disc Herniation Due to Annulus-Endplate Junction Failure: A Mechanical and Microstructural Investigation.

STUDY DESIGN: Microstructural investigation of compression-induced herniation of the flexed lumbar disc. OBJECTIVE: To provide a microstructural analysis of the mechanisms of annular wall failure in healthy discs subjected to flexion and a rate of compression comparable with the maximum rate at which the muscles of the spinal column can generate a force. SUMMARY OF BACKGROUND DATA: Clinical evidence indicates the involvement of the endplate in herniation. It is known that both an elevated rate of compression and a flexed posture are necessary to cause disc failure either within the midspan of the annulus or at the annular-endplate interface. However, the question of what effect a sudden or "surprise" loading might have on the mode of failure is, as yet, unanswered. METHODS: Twenty-four healthy mature ovine lumbar motion segments were compressed to failure in high physiological flexion (10º). This occurred over approximately 5 mm of crosshead displacement in 0.75 seconds that resulted in a displacement rate of 400 mm/min (defined as a "surprise" rate) and was intended to simulate the maximum rate at which the muscles of the spinal column can generate a force. The damaged discs were then analyzed microstructurally. RESULTS: Fifty-eight percent of discs suffered annular-endplate junction rupture, 25% suffered midspan annular rupture, and the balance of 17% endplate fracture. Microstructural analysis indicated that annular rupture initiated at the endplate apical ridge in the mid-to-outer region of the annulus in both annular-endplate and midspan annulus rupture. CONCLUSION: Motion segments subjected to a "surprise" loading rate are likely to fail via some form of annular rupture. Failure under such sudden loading occurs mostly via rupture of the annular-endplate junction and is thought to arise from a rate-induced mechanostructural imbalance between the annulus and the endplate. LEVEL OF EVIDENCE: N/A.

Crimp morphology in the ovine anterior cruciate ligament.

While the crimp morphology in ligaments and tendons has been described in detail in the literature, its relative distribution within the tissue has not been studied, especially in relation to the complex multi-bundle arrangement as is found in the anterior cruciate ligament (ACL). In this study, the crimp morphology of the ovine ACL was examined topologically and with respect to its double-bundle structure. The crimp morphologies were compared with the knee in three knee positions, namely stance, maximum extension and maximum flexion. As a control, the crimp morphology of the ACL free from its bony attachments was determined. In the control samples, the anterior-medial (AM) bundle contained a combination of coarse and fine crimp, whereas the posterior-lateral (PL) bundle manifested only a coarse crimp. Using the extent of crimp loss observed when subjecting the knee to the respective positions, and comparing with the controls, the crimp morphologies show that the AM bundle of the ACL is most active in the stance position, whereas for the maximum extension and flexion positions the PL bundle is most active. We propose that these differences in crimp morphologies have relevance to ACL design and function.