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A simple and environmentally friendly approach toward the thermoplastic processing of rapidly degradable plastic-enzyme composites using three-dimensional (3D) printing techniques is described. Polycaprolactone/Amano lipase (PCL/AL) composite films (10 mm × 10 mm; height [ h ] = ∼400 μm) with an AL loading of 0.1, 1.0, and 5.0% were prepared via 3D printing techniques that entail direct mixing in the solid state and thermal layer-by-layer extrusion. It was found that AL can tolerate in situ processing temperatures up to 130 °C in the solid-state for 60 min without loss of enzymatic activity. The composites were degraded in phosphate buffer (8 mg/mL, composite to buffer) for 7 days at 37 °C and the resulting average percent total weight loss (WL avg % ) was found to be 5.2, 92.9, and 100%, for the 0.1, 1.0, and 5.0% films, respectively. The degradation rates of PCL/AL composites were found to be faster than AL applied externally in the buffer. Thicker PCL/AL 1.0% films (10 mm × 10 mm; h = ∼500 μm) were also degraded over a 7 day period to examine how the weight loss occurs over time with 3.0, 18.1, 36.4, 46.4, and 70.2% weight loss for days 1, 2, 3, 4, and 7, respectively. Differential scanning calorimetry (DSC) analysis shows that the film's percent crystallinity ( D xtal% ) increases over time with D xtal% = 46.5 for day 0 and 53.1% for day 7. Scanning electron microscopy (SEM) analysis found that film erosion begins at the surface and that water can penetrate the interior via surface pores activating the enzymes embedded in the film. Controlled release experiments utilizing dye-loaded PCL/AL/dye (AL = 1.0%; dye = 0.1%) composites were degraded over a 7 day period with the bulk of the dye released by the fourth day. The PCL/AL multimaterial objects containing AL-resistant polylactic acid (PLA) were also printed and degraded to demonstrate the application of this material on more complex structures.

The aging western society is heavily afflicted with intervertebral disc (IVD) degeneration. Replacement or repair of the degenerated IVD with an artificial bio-mimetic construct is one of the challenges of future research due to its complex structure and unique biomechanical function. Herein, biocomposite laminates made of long collagen fibers in unidirectional (-1.3 ± 2.1°) and angle-plied ± 30° orientations (30.4 ± 6.4 and -29.8 ± 4.5), embedded in alginate hydrogel, were fabricated to mimic the form of single annulus fibrosus (AF) lamella and the circumferential AF, respectively. The mechanical behavior of the composites was measured and compared with in vitro existing data of the human native AF as well as with new data obtained from ovine and bovine specimens. The mechanical behavior was found to reproduce the full stress- strain behavior of the human AF single lamella in several regions of the AF and the Young's modulus was 28.3 ± 8.6 MPa. Moreover, the modulus of the angle-plied laminates was 16.8 ± 2.9 MPa, which is approximately 5% less than the in vitro data. The full stress-strain behavior was also compared with bovine and ovine circumferential AF samples and found to be very similar, with a difference in the modulus of 4.1% and 19.7%, respectively. Moreover, an FE model of the L3-L4 functional spinal unit (FSU) was developed and calibrated to evaluate the mechanical ability of the biocomposite to be used as an AF substitute under physiological IVD loading modes. The biocomposite demonstrated a good ability to mimic the stiffness of the native tissue under physiologic loading modes as flexion, extension, lateral bending and compression, but was too flexible under torsion. It was found that the proposed biomimetics AF design resulted in a compatible function in several mechanical levels, which holds great potential to be used as a viable AF replacement towards full IVD engineering., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The annulus fibrosus (AF) of the intervertebral disc (IVD) consists of a set of concentric layers composed of a primary circumferential collagen fibers arranged in an alternating oblique orientation. Moreover, there exists an additional secondary set of radial translamellar collagen fibers which connects the concentric layers, creating an interconnected fiber network. The aim of this study was to investigate the mechanical role of the radial fiber network. Toward that goal, a three-dimensional (3D) finite element model of the L3-L4 spinal segment was generated and calibrated to axial compression and pure moment loading. The AF model explicitly recognizes the two heterogeneous networks of fibers. The presence of radial fibers demonstrated a pronounced effect on the local disc responses under lateral bending, flexion, and extension modes. In these modes, the radial fibers were in a tensile state in the disc region that subjected to compression. In addition, the circumferential fibers, on the opposite side of the IVD, were also under tension. The local stress in the matrix was decreased in up to 9% in the radial fibers presence. This implies an active fiber network acting collectively to reduce the stresses and strains in the AF lamellae. Moreover, a reduction of 26.6% in the matrix sideways expansion was seen in the presence of the radial fibers near the neutral bending axis of the disc. The proposed biomechanical model provided a new insight into the mechanical role of the radial collagen fibers in the AF structure. This model can assist in the design of future IVD substitutes., (Copyright © 2019 by ASME.)

Background: The intervertebral disc (IVD) is a complex organ that acts as a flexible coupling between two adjacent vertebral bodies and must therefore accommodate compression, bending, and torsion. It consists of three main components, which are elegantly structured to allow this: the annulus fibrosus (AF), the nucleus pulposus (NP), and the end-plates (EP)., Purpose: Thus far, it has not been possible to examine the microarchitecture of the disc directly in three dimensions in its unaltered state and thus knowledge of the overall architecture of the disc has been inferred from a range of imaging sources, or by using destructive methods., Study Design: A nondestructive ultrahigh field Magnetic Resonance Imaging (MRI) of 11.7 T was used together with image analysis to visualize the ovine IVDs., Methods: Three-dimensional image stacks from eight IVDs harvested from sheep, half of which were 4 to 5 years old and the others approximately 2 years old were reconstructed and examined, and their microstructure were imaged. The overall structure of the disc, including the average of 14 AF lamellae (9-28), NP, and EP was then visualized with particular attention given to integrating elements as radial translamellar cross-links, AF-NP transition zone EP-AF integration and EP-NP insertion nodes (ie the connecting junctions between the EP and NP). Moreover, collagen fiber orientation was determined at different depths and locations throughout the annulus., Results: It was found that there was a clearer demarcation in the AF-NP transition zone of the younger discs compared with the older ones. This difference was reflected in the visibility of AF-NP and EP-AF integration. It was also possible to view the fiber architecture of the AF-NP integration in greater depth than was possible previously with histological techniques. These fibers were mainly observed in the younger discs and their length was measured to be of 2.6 ± 0.2 mm., Conclusions: The present results provide a substantial advance in visualization of the three-dimensional architecture of an intact IVD and the integration of its components., (Copyright © 2018. Published by Elsevier Inc.)

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.

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.

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.

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.

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 ± 500 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.

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.

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.

Study Design: Microstructural investigation of compression-induced disruption 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 an elevated rate of compression., Summary of Background Data: At the level of the motion segment failure of the disc in compression has been extensively studied. However, at the microstructural level the exact mechanisms of disc failure are still poorly understood, especially in relation to loading posture and rate., Methods: Seventy-two healthy mature ovine lumbar motion segments were compressed to failure in either a neutral posture or in high physiological flexion (10°) at a displacement rate of either 2 mm/min (low) or 40 mm/min (high). Testing at the high rate was terminated at stages ranging from initial wall tearing through to facet fracture so as to capture the evolution of failure up to full herniation. The damaged discs were then analyzed microstructurally., Results: Approximately, 50% of the motion segments compressed in flexion at the high rate experienced annulus or annulus-endplate junction failure, the remainder failed via endplate fracture with no detectable wall damage. The average load to induce disc failure in flexion was 18% lower (P < 0.05) than that required to induce endplate fracture. Microstructural analysis indicated that wall rupture occurred first in the posterior mid-then-outer annulus., Conclusion: Disc wall failure in healthy motion segments requires both flexion and an elevated rate of compression. Damage is initiated in the mid-then-outer annular fibers, this a likely consequence of the higher strain burden in these same fibers arising from endplate curvature. Given the similarity in geometry between ovine and human endplates, it is proposed that comparable mechanisms of damage initiation and herniation occur in human lumbar discs., Level of Evidence: N/A.

Purpose: Recent investigations using an ovine spine model have established that the disc nucleus contains a highly convoluted fibre network with endplate-to-endplate connectivity, this connectivity being achieved via distinctive nodal attachment points. The purpose of this study was to investigate how this nodal anchoring system might be influenced by maturation., Methods: Lumbar motion segments were dissected from newborn, 3, 12 months and fully mature ovine animals, subjected to a novel annular ring-severing procedure to remove the strain-limiting influence of the annulus, then either mechanically tested to destruction or examined microstructurally and ultrastructurally. The morphology of the nodes and their linear density within the relatively thin section planes were analysed to provide a basis for comparison between the four age groups., Results: Mechanical testing following ring severing revealed that the remaining nuclear material in all samples, irrespective of maturity, had the ability to transmit a substantial load from endplate to endplate. Imaging of the ring-severed samples from all age groups in their stretched, but unruptured state revealed the presence of axially aligned fibrosity in the nucleus region consistent with endplate-to-endplate connectivity. Endplate insertion nodes were observed in all age groups. Ultrastructural examination revealed that the fibrillar architecture of these nodes in the newborn discs was similar to that observed in the nodes of mature discs. However, there was a rapid increase in their linear density between birth and 3 months, after which this remained constant., Conclusions: The nodal attachment points identified previously in mature ovine discs are also present in newborn, and 3- and 12-month-old animals with an initial rapid increase in their linear density between birth and 3 months, after which it remained constant. The size and morphology of the attachment points were similar for all ages. Our study suggests that the increase in nodal density in the ovine disc endplate is part of an adaptive response to the loading environment that the disc is exposed to from birth to maturity.

Study Design: Mechanical and microstructural assessment of nucleus-annulus integration., Objective: To investigate the existence of structural integration between the nucleus and the inner annulus., Summary of Background Data: The nucleus is often viewed as a hydrostatically functioning entity that is largely separate from its surroundings. The boundary between nucleus and annulus is acknowledged as difficult to define., Methods: Ten-millimeter-thick sagittal slabs were cut from the central region of ovine lumbar discs. The annulus-nucleus transition region was isolated and the resulting samples subjected to transverse tensile loading up to failure. Similar samples were stretched to about 4 to 5 times their original separation and then subjected to microstructural examination to investigate structural integration across the inner annulus-nucleus region., Results: The annulus-nucleus boundary could support an average load of 5.7 N (range, 2-11.5 N). Tensile loading causes the fibrous structure of the nucleus to be drawn into an approximate alignment in the transverse stretch direction with an associated reverse inpulling of the inner annular layers. At high magnification, the horizontally aligned nucleus fibers can be seen to branch and blend with the inner annular structure., Conclusion: The nucleus contains a convoluted but highly structured network of fibers of varying length, which appear to integrate with the inner annulus and confer a significant degree of transverse interconnectivity that can be demonstrated mechanically. This new experimental evidence, together with that from a previous study demonstrating nucleus-endplate connectivity, makes it clear that the nucleus cannot be considered as a separate entity in the disc. We propose that this structural integration provides the nucleus with a form of tethered mobility that supports physiological functions distinct from the primary strength requirements of the motion segment.

The intervertebral disc nucleus has traditionally been viewed as a largely unstructured amorphous gel having little obvious integration with the cartilaginous endplates (CEPs). However, recent work by the present authors has provided clear evidence of structural cohesion across the nucleus-endplate junction via a distinctive microanatomical feature termed insertion nodes. The aim of this study was to explore the nature of these insertion nodes at the fibrillar level. Specially prepared vertebra-nucleus-vertebra composite samples from ovine lumbar motion segments were extended axially and chemically fixed in this stretched state, and then decalcified. Sections taken from the samples were prepared for examination by scanning electron microscopy. A close morphological correlation was obtained between previously published optical microscopic images of the nodes and those seen using low magnification SEM. Progressively high magnifications provided insight into the fibrillar-level modes of structural integration across the nucleus-endplate junction. The closely packed fibrils of the CEP were largely parallel to the vertebral endplate and formed a dense, multi-layer substrate within which the nodal fibrils appeared to be anchored. Our idealised structural model proposes a mechanism by which this integration is achieved. The nodal fibrils, in curving into the CEP, are locked in place within its close-packed layers of transversely aligned fibrils, and probably at multiple levels. Secondly, there appears to be a subtle interweaving of the strongly aligned nodal fibrils with the multi-directional endplate fibrils. It is suggested that this structural integration provides the nucleus with a form of tethered mobility that supports physiological functions quite distinct from the primary strength requirements of the disc., (© 2012 The Authors. Journal of Anatomy © 2012 Anatomical Society.)

Background Context: The intervertebral disc plays a major functional role in the spinal column, providing jointed flexibility and force transmission. The end plate acts as an important structural transition between the hard vertebral tissues and the compliant disc tissues and is therefore a region of potentially high stress concentration. The effectiveness of anchorage of the tough annulus fibers in the end plate will have a major influence on the overall strength of the motion segment. Failure of the end plate region is known to be associated with disc herniation., Purpose: The aim of this study was to investigate the mechanism of anchorage of the annular fibers in the end plate., Study Design: A microstructural analysis of the annulus-end plate region was carried out using motion segments obtained from the lumbar spines of mature ovine animals., Methods: Motion segments were fixed and then decalcified. Samples incorporating the posterior annulus-end plate were then removed and cryosectioned along the plane of one of the lamellar fiber directions to obtain oblique interlamellar sections. These sections were imaged in their fully hydrated state using differential interference contrast optical microscopy., Results: The annular fiber bundles on entering the end plate are shown to subdivide into subbundles to form a three-dimensional multileaf morphology with each leaf separated by cartilaginous end plate matrix. This branched morphology increases the interface area between bundle and matrix in proportion to the number of subbundles formed., Conclusions: Given both the limited thickness of the end plate and the intrinsic strength of the interface bond between bundle and end plate matrix, the branched morphology is consistent with a mechanism of optimal shear stress transfer wherein a greater strength of annular fiber anchorage can be achieved over a relatively short insertion distance., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The disc nucleus is commonly thought of as a largely unstructured gel. However, exactly how the nucleus integrates structurally with the endplates remains somewhat ambiguous. The purpose of this study was to investigate whether a substantial level of structural/mechanical cohesion does, in fact, exist across the nucleus-endplate junction. Vertebra-nucleus-vertebra samples were obtained from mature ovine lumbar motion segments and subjected to a novel technique involving circumferential transverse severing (i.e. ring-severing) of the annulus fibrosus designed to eliminate its strain-limiting influence. These samples were loaded in tension and then chemically fixed in order to preserve the stretched nucleus material. Structural continuity across the nucleus-endplate junctions was sufficient for the samples to support, on average, 20 N before tensile failure occurred. Microscopic examination revealed nucleus fibres inserting into the endplates and the significant level of load carried by the nucleus material indicates that there is some form of structural continuity from vertebra to vertebra in the central nucleus region.