A fresh look at the nucleus-endplate region: new evidence for significant structural integration
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- Wade, K.R., Robertson, P.A. & Broom, N.D. Eur Spine J (2011) 20: 1225. doi:10.1007/s00586-011-1704-y
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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.
KeywordsDisc nucleusCartilaginous endplateFibrosityTethering mechanisms
The disc, consisting of three main components—the nucleus, the annulus, and the cartilaginous endplates—provides a strong but flexible linkage between adjacent vertebrae. The nucleus is surrounded by the concentric annular layers or lamellae with each comprising parallel arrays of collagen fibres crossing obliquely at alternating angles between adjacent layers. The annulus and nucleus are contained superiorly and inferiorly between the cartilaginous endplates, the latter, in turn, structurally linked to the vertebral bodies via the vertebral endplates. Under compression, the nucleus, enclosed by the annulus, is loaded hydrostatically, and by expanding against the walls of the annulus, transfers compressive loads into the annulus [1–8].
The annulus has been extensively studied by many researchers. Tensile testing has been used to determine the bulk mechanical properties of the annular wall. However, the highly directional arrangement of fibres within each layer means that considerable care must be taken to ensure that test samples are prepared such that fibre orientation incorporation within the sample does actually reflect the true strength contribution of these fibres [9–12].
Our own research group has used a variety of combined micromechanical and microstructural techniques to investigate the subtler aspects of disc microanatomy and its functional implications. By stretching carefully orientated sections of fully hydrated annulus either across or along the primary in-plane fibre direction and simultaneously viewing microstructural responses, a number of important structural features in the disc have now been identified. Specifically, both fibrillar interactions within and between layers have been demonstrated and an extensive interlamellar bridging network has been identified which has implications for explaining interlamellar cohesion in the annulus and hence its mechanical properties [13–16].
In contrast, little consideration seems to have been given to the extent to which the nucleus is integrated with the endplates or even the annulus. Although much of the literature seems to suggest that the non-degenerate nucleus is largely an amorphous, gelatinous structure, several researchers have identified a sparse network of elastin fibres within the nucleus [17–19]. Some ultrastructural studies have concluded that there is no significant attachment between the nucleus and the cartilaginous endplate [20–22]. Other authors suggest that while there is some connection between the nucleus fibres and the cartilaginous endplate, it is via a poorly organised structure . Another view is that the nucleus is a much smaller mid-disc-height structure that is not connected to the cartilaginous endplates at all. Rather, the nucleus is totally contained by the inner annulus fibres which sweep around it and insert directly into the central endplate region [24, 25].
With the above structural ambiguities in mind, the primary aim of this new investigation was to explore the nature of any structural relationship between the nucleus and the cartilaginous endplates.
An assessment of the tensile properties of the extracted vertebra–nucleus–vertebra sample was conducted on 12 samples from 4 spines using the following procedure: each sample was mounted between the plinths of an Instron 5543 testing machine fitted with a 1,000-N load cell. Tissue glue was used for this mounting procedure whilst ensuring that the sample was not subjected to any bending moment, i.e. only an axial tensile load was applied. All 12 tests were carried out with a crosshead displacement rate of 0.5 mm/min. Loading was terminated at approximately 30–40 N, due to the limitations of the sample attachment method; this resulted in axial extensions within the disc tissue of no more than ~1.5 mm. Each sample was kept hydrated with physiological saline throughout the testing procedure.
The sample was unloaded and then temporarily detached from the testing machine whilst retaining the ability to relocate it in exactly the same position for further testing. The residual annular elements were then carefully and progressively ring-severed, their complete severance indicated by a sudden, large increase in both extension and mobility of the remaining nucleus material. The ring-severed sample was then remounted in the testing machine and retested at the same displacement rate used previously. All tests on these 12 ring-severed samples were taken to near failure. The results from the tensile testing were presented as raw load–displacement curves (as opposed to stress–strain curves) due to the difficulties of obtaining an accurate measurement of the dimensions of the ring-severed sample, especially during the later stages of the test. Employing this procedure meant that accurate comparisons of response were confined to the unsevered versus severed states within each sample, rather than between samples.
For the microstructural studies, a further 11 vertebra–nucleus–vertebra samples were subjected to ring-severing only, and then manually stretched, followed by chemical fixation in their stretched state, and finally decalcified. From prior experimentation it was found that the remaining nucleus mass could be extended up to five times its original axial height (i.e. up to 500% strain) before reaching a limiting extension beyond which progressive failure occurred (see Fig. 2b). Each of the 11 ring-severed samples was held in this highly stretched but unruptured state by applying a minor load of approximately 1 N, sufficient to maintain the nucleus material in its near strain-limiting state (see Fig. 2b) followed by fixation for 3 days in 10% formalin and decalcification for 14 days in 10% formic acid.
All samples for microstructural analysis were then appropriately trimmed and 30-μm thick sagittal sections obtained by cryosectioning. These sections were then wet-mounted on slides and examined using differential interference contrast optical microscopy (DIC). In addition, to obtain clearer differentiation between the nucleus and inner annulus, three intact motion segments were fixed, decalcified, and sectioned sagittally, and then examined using DIC. A total of 26 motion segments from 13 ovine lumbar spines were used in the study.
As mentioned in our introduction the current literature presents a number of contrasting views on the extent and manner of integration of the nucleus with the endplate [20–25]. We believe this absence of a structural consensus has arisen because of the lack of a suitable experimental method to remove the influence of the annulus on nucleus unravelling. The novel ring-severing technique employed in this new study has enabled us to examine more critically the nature of nucleus-endplate integration.
It should be noted that although the transition between outer nucleus and inner annulus is structurally somewhat vague, our study employed two distinct discriminating criteria that enabled us to define the remaining material as essentially nucleus. First, the sudden large extension observed following ring-severing confirmed the functional dislocation of all fibres that had contributed to the strain-restricting properties of the unsevered annulus arising from its cross-ply lamellar structure. Secondly, the obvious morphological difference in endplate insertion morphology between the nucleus and annular regions (c.f. Figs. 8, 11 with Fig. 12) confirmed that the samples in their ring-severed state represented the behaviour of the isolated nucleus.
The ring-severing procedure we have used in this study allows the nucleus as defined above to be loaded directly in tension. The dramatic extension that precedes the point of rapid increase in modulus (see region J on curve B in Fig. 5) is clear confirmation that the nucleus possesses some form of structural continuity, is highly convoluted, is fully integrated with the endplates via two modes of direct fibrous insertion (see Figs. 8, 11), and has an intrinsic tensile strength. Following the phase of easy extension and rapid stiffening (the “J” region in Fig. 4), there is a progressive but irregular rise to the peak load with eventual failure preceded by a gradual though erratic drop in load, and further large extension of the nucleus mass (region K in Fig. 4). This irregular form of the load–displacement curve suggests that the fibres within the nucleus region have varying degrees of convolution (and thus have varying extendable lengths) and hence are being loaded and ruptured sequentially rather than simultaneously.
Our images clearly indicate that nearer the nucleus edge of the stretched ring-severed tissue the fibres have become strongly aligned in the stretching direction (see region in box B in Fig. 5 and enlarged in Fig. 7), thus confirming that there is endplate-to-endplate load transmission by these fibres. This ability to transmit force obviously implies some form of structural continuity of the fibres themselves. In fact this continuity nearer the nucleus edge was readily observed and recorded using multiple image maps (each >50 images), but for practical reasons could not be included in our data presentation. Also, due to the inability of light microscopy to image individual collagen fibrils the present study cannot confirm the exact basis of this structural continuity. Whether fibrils themselves run continuously from endplate to endplate, or whether the fibres are constructed from shorter lengths of overlapping fibrils remains an unresolved question.
That the nucleus has resistance to tension when it clearly operates in compression raises the question as to why it might possess this tensile property. It should be emphasized that we have used tensile stretching to demonstrate the existence of endplate-to-endplate structural/mechanical cohesion. However, during normal disc function in vivo the nucleus fibres will not see this tensile mode of loading. We therefore suggest that the integration of the fibres with the endplates acts to provide the nucleus with a form of tethered mobility. In this way the fibres would function (1) to contain the proteoglycans via some form of collagen–proteoglycan interaction so as to maintain the hydration potential of the nucleus [26, 27], and (2) to provide a substrate for the cells within the nucleus so as to maintain an appropriate biology. The convoluted geometry of the fibres would still confer a high degree of freedom so as to accommodate nucleus shape changes associated with normal disc function in which hydrostatic loading plays an essential role .
In summary, the present study provides new insights into what constitutes a normal nucleus-endplate structural relationship, at least in the mature ovine spine. It offers a structural framework for exploring how early degenerative changes might alter the nucleus-endplate relationship. Our results provide a contrasting view to much of the published literature which either states that there is no significant connection between the nucleus and endplate, or largely ignores the issue. Our experiments, demonstrating that the central nucleus alone is capable of carrying substantial tensile loads, provide clear evidence that it is highly structured, though convoluted, and hence can give the appearance of disorder to the casual observer using standard sectioning planes.
The ovine nucleus possesses a distinct fibrosity which displays structural continuity from endplate to endplate. This fibrosity is structurally integrated with the cartilaginous endplates and is capable of withstanding significant tensile forces. A structured though convoluted, tethered nucleus model is proposed that supports the general concept of a relatively mobile nucleus behaving hydrostatically within the confinement of the annulus.
The authors are grateful for the award of funding in support of this research from both the Wishbone Trust (New Zealand Orthopaedic Association) and the University of Auckland.