Neil D. Broom , PhD, Biomaterials Laboratory, Department of Chemical and Materials Engineering, The University of Auckland, Private Bag 92019, Auckland City, New Zealand. F: + 64 9 3737463; E-mail: firstname.lastname@example.org
Little is known about the complex forces acting on the deformable multi-layered annulus at a microstructural level as the spine is compressed, flexed and twisted. The recently described translamellar bridging network radially linking many lamellae at discrete locations around the disc wall could be expected to play a significant biomechanical role. In this study, segments of annular wall that were sectioned at a range of angles (oblique, in-plane, sagittal and transverse) were examined using differential interference contrast microscopy to fully elucidate the fibrous detail of the translamellar bridging structures. Typically encompassing a width of 300–600 µm, translamellar bridging fibres proceed radially in the interbundle space within an individual lamella. Upon traversing the lamella, the bulk of these radial fibres bend through 90° to merge with the fibres of the adjacent lamellae. The central fibres of this bridging system continue into the equivalent bridging structures in the adjacent lamellae. As well as exposing structural details that underpin the biomechanical properties of the disc wall, this study has also exposed the limitations of using standard section planes commonly employed by disc researchers.
Little is known about the complex forces acting on the deformable multi-layered annulus at a microstructural level as the spine is compressed, flexed and twisted. An insight into some of the interactions involved was revealed in a confocal microscopy study of bovine discs by Bruehlmann et al. (2004), where they reported slip within lamellae in a disc subjected to flexion. Earlier researchers have shown slip between adjacent lamellae accompanies torsion, flexion and extension (Hickey & Hukins, 1980; Klein & Hukins, 1982a). The change in orientation of collagen fibres in the rabbit disc wall due to a 5° torsion (≈ 5× more than the human physiological range; Gregersen & Lucas, 1967) was associated with a 2–3º change in the tilt angle of the fibres (Klein & Hukins, 1982a,b). A similarly small amount of lamellar reorientation was reported by Bruehlmann et al. (2004), with an 8° flexion causing a 3–4° rotation towards the axial direction in the posterior annulus.
To appreciate how the gross mechanical loads on the disc are translated into forces experienced within and between lamellae, a detailed understanding of the disc microstructure is essential. Histological techniques used by Melrose et al. (2008), Yu et al. (2005), Smith & Fazzalari (2006) and others have advanced our understanding of the disc wall microcomponents. However, there remain significant gaps in our understanding of microstructure; the work published by Marchand & Ahmed (1990) and, more recently Pezowicz et al. (2005, 2006) have helped rectify this situation. Marchand & Ahmed peeled dehydrated discs to describe complete and incomplete layers around the wall, fibre angles, and fibre bundle geometry. Pezowicz et al. performed micromechanical experiments on thin hydrated slices exposing relationships within and between lamellae of the disc wall. Extension of the analysis of lamellar interconnectivity to its full 3-dimensional form led to the recently published block model of the translamellar bridging network (TLBN) (Schollum et al. 2008). This was developed using differential interference contrast (DIC) microscopy to analyze sections taken at 100–200-µm intervals obliquely through the annular wall and offered a preliminary description of the extent and form of the translamellar bridges. The study demonstrated the multilamellar nature of the bridges, their close association with the lamellar architecture and their density within the annular wall.
In this new investigation, we have sought to gain further insights into the fibrous organization of the TLBN by using similar techniques, but with additional section angles (oblique, in-plane, sagittal and transverse). This clarification of the interrelationships between the primary load-bearing components constituting the annulus is fundamental to our understanding of disc biomechanics. Additionally, it is both the widely accepted practice of using transverse and sagittal sectioning in disc research and the difficulty of navigating oneself around the complex annular microstructure that would make the rationalization of sagittal and transverse views of the TLBN valuable. There is an unexplained variation in the form of the translamellar bridging structures in published images – often thin and stepped, but at times more direct and thicker and terminating abruptly. In the stepped form, do the bridging elements change position relative to their neighbours? There is, to date, little understanding of how these structures associate with the complex lamellar architecture in a 3-dimensional sense, leaving the investigator unable to compare the bridging structures as viewed in one section plane with those viewed at a different angle.
Five non-degenerate immature lumbar spines from 4–7-month-old ovine animals were collected and kept frozen at –20 °C until required. Analysis was confined to the anterior half of discs L5–6 and L4–5. Motion segments were sawn from the frozen spine and extraneous tissue removed to expose the intervertebral disc. Following fixation and decalcification, four ≈ 40° anterior segments were generated (see Fig. 1A). The nucleus was removed, leaving a thickness of between 3 and 4 mm of outer annulus. The annulus was kept moist with 0.15 m saline throughout. It should be noted that with the aid of a magnifying lens the outer lamella fibre angle was clearly visible in these young discs. The previously used technique (see Pezowicz et al. 2006 and Schollum et al. 2008) of sectioning across the disc face to expose structural relationships between lamellae entailed the use of a fine-tipped black ink pen first to make a series of dots marking the fibre angle. The upper left and lower right corners of the quadrant were then trimmed with a scalpel to either the angle as marked (if a 30° section plane was required), or on a 15° steeper angle (if a 45° section plane was required). With corners trimmed, the blocks were then positioned on an aluminium block with the desired cutting surface roughly horizontal, in a small pool of cryo tissue gel and frozen with liquid nitrogen. Based on the earlier ink marks, final adjustments to the cutting blade angle were made prior to sectioning with a freezing sledging microtome.
For the initial 10 quadrants (taken from spines 1, 2 and 3) 30-µm-thick serial slices were sectioned 45° above horizontal (oblique) (see Fig. 1B). With successive lamellae aligned approximately 30° above and then 30° below the horizontal plane, the 45° cutting plane contained successive lamellae alternately near in-plane (lamellar fibre angle is 15° off parallel to the section angle) and near cross-section (lamellar fibre angle is 15° off perpendicular to the section angle). The serial slices were viewed with DIC microscopy in their fully hydrated state. Examination of the oblique serial slices led to the development of a preliminary 3-dimensional fibrous model of the translamellar bridges. The remaining quadrants (taken from spines 2, 3, 4 and 5) were similarly processed, but at section planes 30°, 90° or 0° above the horizontal (see Fig. 1C–E, respectively) to verify the model.
With a 30° section plane (see Fig. 1C), the slices contained the successive lamellae alternately parallel to the cutting plane (i.e. 0° offset between lamellar fibre angle and cutting-plane) and 30° off the perpendicular to the section plane. As can be seen in Fig. 1D and E, the sagittal and transverse cutting planes were both symmetrical with respect to the lamellar fibre directions. In a sagittal slice (Fig. 1D) the fibre angles of the successive lamellae were offset by 60° to the cutting-plane, i.e. a 30° offset from being perpendicular to the cutting-plane angle. In the case of the sagittal quadrants, only the two most anterior quadrants were used, with the section plane being the vertical plane between these two central quadrants. Lastly, in a transverse slice (Fig. 1E) the fibre angles of the successive lamellae were offset by 30° from parallel to the cutting-plane.
Much of the work presented in this study focused on close examination of small portions of the translamellar bridging network. But looking first at the macro view offered in Fig. 2, clearly the translamellar bridges are significant structures, typically traversing 12+ lamellae in a single 30-µm-thick oblique slice. As described previously (Schollum et al. 2008), the immature anterior ovine annulus usually contains one or two of these core structures per 20° quadrant. They could therefore be expected to play a significant biomechanical role. All discs examined in this study fitted within these descriptive parameters and, importantly, there were consistent characteristics for each of the 30°, 90° and 0° section planes as examined across four spines at two disc levels, and for the 45° section plane across three spines at two disc levels.
Consider first the slices of annulus in Fig. 3, obtained using a 45° section plane. The alternating lamellae are exposed as near in-plane (nIP) and near cross-section (nCS). In Fig. 3A a substantial and characteristically serpentine translamellar bridging structure radially connects nine lamellae. A high-magnification image in Fig. 3B reveals differences in the fibrous detail of the bridging elements as they traverse near in-plane and near cross-section lamellae; The near cross-section lamellae (see nCS in Fig. 3B, and a further example at a higher magnification in Fig. 3C) are traversed in a direct path by predominantly complete crimped fibres (see white arrow Fig. 3B, and white wavy line Fig. 3C). The bridging element broadens as the fibres merge with the lamellar fibres of the adjacent near in-plane lamellae.
Near cross-section traverses are commonly present in 10–20 serial slices, indicating a structure persisting for 300–600 µm in the interbundle space of a lamella, radially linking the adjacent lamellae with the same fibre orientation. In contrast, even though the bridging structure also traverses the near in-plane lamellae (see nIP in Fig. 3B), the fibres it is composed of appear to be only partially captured in this 30-µm section depth (see black arrow). This suggests we need to look beyond a single slice. The series of five micrographs in Fig. 4A–E tracks a single near in-plane traverse through 120 µm of neighbouring serial sections. The centre of the series is shown in Fig. 4C, cropped from the larger image in Fig. 3A. The serial slice 30 µm immediately above, in Fig. 4B, shows the bridging fibres traversing near in-plane lamella L4 further to the right. The same traverse 30 µm immediately below, in Fig. 4D, has the bridging fibres traversing this same lamella, L4, further to the left. In Fig. 4A and E the bridging is faintly visible (see black arrows) further to the right and left, respectively. In the sections above Fig. 4A and below Fig. 4E the traverse of near in-plane lamella L4 was no longer visible (not shown), which would allow a 150-µm step to overlook this connection, and even at a 90-µm interval the significance of the link could be missed.
The series Fig. 4A–E therefore demonstrates that with a 45° section plane, the near in-plane traverse is gradually revealed in subsequent slices, with five 30-µm micrographs necessary (in this typical case) to fully encompass the traverse of near in-plane lamella L4. A single micrograph conveys an incorrect impression of the width of the structure. In fact, the distance between the black arrowheads in Fig. 4A and E indicates a ≈ 300-µm width of bridging fibres, which is of equal persistence to those traverses observed in near cross-section lamellae, such as L3. This has enabled us to propose that the translamellar bridging fibres are laid similarly in the interbundle space of adjacent lamellae, forming a criss-cross structure with the subsequent bridge. This is described schematically in Fig. 5 positioning the proposed bridging structure in a portion of annular wall. Figure 5A shows a front elevation of a quadrant of annular wall with attached vertebrae, and the outermost lamella removed. From this wall has been taken a small block (Fig. 5B). The orientation of Fig. 5B is best understood by visualizing the front elevation in Fig. 5A tilted backwards, with the vertebrae now lying at the front and back, and the outermost lamella (L2 in this case) forming the top of the block. For simplicity the small block encompasses only two lamellae (L2 and L3), subsequent lamellae closer to the nucleus could be imagined stacked beneath the two drawn. These two lamellae each comprise two bundles of collagen fibres, with bridging network fibres enclosed in the structure.
In Fig. 5C, the lamella L2 has been erased schematically, exposing two bridging elements (B2 and B3). B2 fibres are constrained to the interbundle space of lamella L2 and are anchored into the lamellae above and below L1 (not shown) and L3. Similarly B3 fibres, confined to the interbundle space of L3, merge with lamellae L2 and L4 (not shown). The aforementioned fibre interactions are indicated in Fig. 5D. Additionally, X marks the site of B2 and B3 bridging fibres continuing radially through the wall.
Figure 5E offers a bird's-eye view of Fig. 5C and D with all lamellar material deleted (effectively taking us back to a front elevation like Fig. 5A, but this time of a small area enlarged and with no lamellar fibres). The bridging fibres are represented by dots in this schematic as they are visible predominantly in cross-section. Each bridging element is entrapped in the interbundle spaces, forming a criss-cross as you move towards the centre of the disc, with B3 located behind B2. Figure 5F is a stylized perspective view of five bridging elements tracking radially towards the centre of the disc, representing just one third of a core-bridging structure such as that shown in Fig. 2.
Testing model validity
To test the validity of this structural model a series of ‘expected’ micrographs generated from the proposed model at each of the section angles shown in Fig. 1B–E (45°, 30°, 90° and 0°) can now be placed alongside micrographs collected experimentally. Aside from model confirmation, this systematic examination of the annular wall also allows exploration of the idiosyncrasies of each of these section types.
45° Section plane (oblique)
Orientation of the 45° section plane with respect to the lamellar structures is shown in Fig. 6A. The lamellar fibres of L2 and L3 will be sectioned with a 15° offset from in-plane and perpendicular, respectively. Clearly, initial micrographs will show only bridge B3 (sited within lamella L3) broadly anchored into the lamellae above and below it. Only close to the intersection of bridging elements B2 and B3 will the two bridges be captured in a single micrograph. The series of micrographs in Fig. 6B contains this intersection of bridging elements in five 30-µm steps, both for the model (shown in schematics) and experimentally (shown in micrographs). In both series the micrograph associated with the section plane passing closest to the centre of the structure is sited on the 3rd row of micrographs, labelled X. The sections above and below X are indicated by +1, +2, –1, –2. Each interval is ≈ 30 µm, encompassing 120 µm in total. The expected and experimental series of micrographs both show bridge B2 first evident curving substantially out to the right (Fig. 6B, X + 2), then to a lesser amount (Fig. 6B, X + 1) and finally in line with bridge B3 (Fig. 6B, X) followed by a similar curvature out to the left as the section plane moves further back (Fig. 6B, X − 1, X − 2). The curved path of the fibres forming the outer surfaces of bridge B2 (see the 3D schematic in Fig. 6A) limits the possibility of complete fibre capture in a flat section. Therefore, apart from the micrograph which encompasses site X, the bridging fibres traversing L2 will most often be truncated by sectioning. The less distinct appearance of the bridging element away from site X, as seen in micrographs Fig. 6B, X + 2 and X − 2, is to be expected, based on the tapered profile shown in the proposed fibrous model (see Fig. 5).
30° Section plane (fibre angle)
Figure 7 offers a similar comparison of expected to actual micrographs but this time with a 30° section plane. As shown in Fig. 7A, this section plane lies parallel to bridge B2 and the fibres in lamella L2, but at a 30° offset to the fibres of cross-sectional lamella L3. It should be noted that the tapering profile of bridge B2 at its extremities will make them less distinct when attempting to view them in the plane of the section. Additionally, the similarity in section and bridging element thickness and the often non-perpendicular structures in the tissue makes entire bridging element capture in this plane unlikely. This understood, there is good correlation between the expected and actual series of micrographs contained in Fig. 7B. At 60 µm before ‘X’ only an isolated bridge B3 in lamella L3 is present (Fig. 7B, X + 2). Bridge B2 is visible only fleetingly, clear for one 30-µm section with a 300-µm + width of in-plane bridging fibres orientated predominantly radially (Fig. 7B, X). The micrographs adjacent to micrograph X (Fig. 7B, X + 1 and X – 1) catch only the splayed bottom and top edges of the bridging element B2 as it anchors into lamellae L3 and L1 (not shown). The non in-plane lamella (L3) is continuously bridged through 120 µm (and beyond, not illustrated here). This series confirms that a thin but wide radial traverse of the in-plane lamella is made by the bridging fibres, demonstrating that the serpentine profile seen in the oblique serial slices (see Figs 2 and 3A) is an artefact of that section angle.
Figure 8 contains some typical micrographs obtained with a 30° section plane, which lend further support to the proposed model. At high magnification the crimped fibres of the in-plane lamella (‘IP’ in Fig. 8B) are traversed by bridging fibres laid predominantly in the radial direction. The bridging fibres are also crimped, indicating alignment with the section plane. Figure 8A is an example of the frequently exclusive presence of bridging elements in the non in-plane lamellae at this section angle, with the bridging elements often aligned radially in these alternate lamellae as you look across the wall (see white arrows). The continuous nature of the translamellar bridging network will only infrequently be exposed. Fig. 8D is an example of one of these events, with a substantial bridge caught traversing an in-plane lamella (IP), and linking into multiple traverses of the neighbouring lamellae. Another feature distinctive to this section angle is the occurrence of dark edges to some in-plane lamellae as shown in Fig. 8C. This occurs in the slices near a principal traverse (as shown in Fig. 7B, X + 1 and X – 1), and is caused by the capture of the wider anchorage.
90° Section plane (sagittal)
Figure 9 offers the comparison of expected and actual micrographs at a section plane 90° above horizontal, or a sagittal section. Orientation of the section plane with respect to the annular structures is shown in Fig. 9A. The section plane fragments the lamellar fibres due to a 30° offset from perpendicular in both lamellae, making the fibrosity of the lamellae indistinct. This is evident in Fig. 10, a typical sagittal section, incorporating seven lamellae and some bridging elements. Here the radial orientation of the bridging fibres can be appreciated but any relationships between the bridging fibres and the lamellae are difficult to discern.
The expected micrographs in Fig. 9B based on the proposed model predict that the initial slice will show bridges B2 and B3 offset from each other, with bridge B2 to the right of bridge B3 (Fig. 9B, X + 2). In subsequent slices (Fig. 9B, X + 1) they will move closer to each other, possibly with signs of some linking fibres, and then finally in-line (Fig. 9B, X) before moving off in opposite directions, with bridge B2 now to the left of bridge B3 (Fig. 9B, X + 1, X + 2). It will take the greatest number of slices to traverse the structure from this angle.
The actual series of micrographs in Fig. 9B track the bridging elements from the lower two lamellae (L6 and L7) in Fig. 10 at 60-µm intervals. These two bridging elements, although not always clearly linked, can be viewed continuously for 240 µm in both lamellae. And, consistent with the proposed model, their position relative to one another changes as you move through the wall. The topmost bridging element is initially positioned to the right of the lower one, and as you move sagitally through the wall its position slides to the left. The progression through the structure takes relatively more slices than was seen in the oblique series (Fig. 6B, at 120 µm), which again concurs with the scenario presented in the proposed fibrous model (see Fig. 5).
0° Section plane (transverse)
Lastly, in Fig. 11 is a comparison of expected and actual micrographs at a section plane 0° above horizontal, or a transverse section. The orientation of this section plane with respect to the lamellar structures is illustrated in Fig. 11A. The fibres of both lamellae are sliced at 30° off the in-plane angle, producing a less fragmented appearance than a sagittal section, but any associations between lamellae and bridging elements will still be unclear. This can be seen in Fig. 12A, with the presence of fibre interactions between these offset bridging elements (white arrows) but no obvious association with the lamellae at this magnification. As typically seen in transverse sections, the bridging elements appear isolated, but with some alignment across alternate lamellae.
The expected micrographs generated from the proposed model in Fig. 11B predict that in a manner similar to the sagittal view, the initial slices will show bridges B2 and B3 offset from one another, but in this case the distance will be even greater (Fig. 11B, X + 2). Each 30-µm step will correspond to a substantial movement in the relative position of bridges B2 and B3. At the point of intersection (Fig. 11B, X) it will be possible to see a very broad bridging structure. Compared to the sagittal view, these bridging elements will appear wider and less distinct, as the section plane meets the bridging structure at a less perpendicular angle, effectively decreasing the fibre density. The actual micrographs in Fig. 11B track a bridging structure comprising two bridging elements in adjacent lamellae through five 30-µm transverse steps. As predicted, these bridging elements move relative to each other in a similar, but more extreme, manner to that just described for sagittal sections. Also, the bridging elements themselves are wider and more blurred than in earlier section angles, consistent with a lower bridging fibre density from a flatter sectioning angle.
Radial but truncated bridging element fibres were typical in the transverse sections when viewed at high magnification. This is consistent with the tendency for the radial bridging system, as it traverses many lamellae, to progress either slightly inferiorly or superiorly through the wall (as shown in the sagittal section in Fig. 12B). This non-horizontal tracking of the TLBN across many lamellae decreases the likelihood of one 30-µm transverse slice encompassing more than a few continuous bridging elements.
By examining the TLBN from multiple directions we can now appreciate the stepped and apparently disconnected bridging elements presented in sagittal and transverse images, but most importantly, this work has allowed a much more rigorous interpretation of the TLBN fibrous architecture. There is an overriding radial orientation of translamellar bridging network fibres through the wall, presumably with a key role in preventing delamination. Based on the fibrous detail garnered in this new study, the schematics in Fig. 13A–C indicate some design characteristics of the annular wall, including means to accommodate small amounts of interlamellar movement expected in normal function (up to 4°; Bruehlmann et al. 2004; Klein & Hukins, 1982a,b);
1Strength of attachments: In Fig. 13A the broad profile of the bridging elements anchored into the lamellae can be seen as they criss-cross through the wall. The central continuous radial link (at X) is accompanied by often 300–600 µm of near-neighbour lamellar anchorage, minimizing the potential of lamellar damage due to a stress concentration. The wrapping of these bridging fibres through two 90° bends as they merge into the lamellae above and below will increase resistance to ‘pull out’ as radial load is applied.
2Independence of neighbouring lamellae: Alternate lamellae, such as L2 and L4 in Fig. 13B, are exposed to near-parallel strains, negating the requirement for slip between them. Bridge B3, lashing together these two lamellae, lies in the gap between collagen bundles of Lamella L3; thus tying Lamellae L2 and L4 together independently of Lamella L3. We therefore have a fibrous organization that keeps the annular wall secured radially, whilst neighbouring lamellae, coursing in opposite oblique directions, are not tightly constrained in the circumferential plane – a feature suited to the variable loading of lamellae with lamellae under tension sometimes occurring adjacent to ones that are relaxed.
3Rotation: Fig. 13C shows how the continuation of the radial bridging fibres at site X (and beyond) generates a central pivot to the structure – this time allowing small amounts of rotation, whilst maintaining a radial link. This engenders the ability to cope with combinations of loads (people twisting whilst lifting an object).
The frequency of these bridging structures will clearly influence how much interlamellar movement is possible. At the expense of reduced flexibility, lamellae firmly locked radially would be less susceptible to inwards buckling if subjected to compression. It has been proposed that the TLBN is a functional adaptation to changing biomechanics in the disc wall (Melrose et al. 2008). In our earlier paper we described the TLBN network becoming more established with age (Schollum et al. 2008). In addition, Melrose et al. noted that the TLBN was most prominent in the mid-annulus, extending only to the outer annular wall with age. This would suggest increased ease of lamellar sliding in the outer wall of the young disc – particularly affecting torsion and bending movements, which predominantly load the outer lamellae (Klein & Hukins, 1983). Thus, in adapting to the effects of an ageing nucleus by more firmly locking the lamellae together via the TLBN, the outer wall loses some of its torsion and bending function.
Published images of the translamellar bridging network as viewed sagitally and transversely in both bovine caudal and ovine lumbar discs (Yu et al. 2002, 2007; Melrose et al. 2008) appear consistent with those presented in this study. With respect to the human model, Yu et al. (2005, 2007) and Smith & Fazzalari (2006) have both described elastin organization in the human disc consistent with bridging elements in the inter-lamellar spaces. But to date there are no published images of their microstructure, leaving us only able to speculate as to their form in the human disc.
To conclude: this study has provided further insights into the fibrous architecture of the disc wall and has allowed rationalization of sagittal and transverse views of the translamellar bridging system. Additionally, the techniques used in this work have highlighted the need for caution when relying on thin histological sections alone. Such sections can provide no more than a limited, abstracted view of the disc's complex 3-dimensional microarchitecture.
The first author acknowledges the award of a University of Auckland Doctoral Scholarship.