Ultrastructural organization of elastic fibers in the septal boundaries of the annulus fibrosus of the intervertebral disc. Please cite this article as: Tavakoli, J., Costi, J.J., Ultrastructural organization of elastic fibers in the partition boundaries of the annulus fibrosus of the intervertebral disc, Acta Biomaterialia (2017), doi: https://doi.org/. However, the clinical relevance of elastic fibers in the annulus fibrosus (AF) of the disc is poorly understood.
Ultrastructural visualization of elastic fibers is an important step towards understanding their structure and function. We compared the visualization of elastic fibers in PB in control and partially digested (digested) samples and discussed their orientation in two different cutting planes (transverse and oblique). The role of elastic fibers in the intervertebral disc is not clearly understood, although few studies have revealed their contribution to the radial integrity of the annulus fibrosus (AF) [5-7].
Several other investigators have confirmed the presence of elastic fibers in the disc; however, their observations did not reveal elastic fiber ultrastructure [ 16 – 19 ]. A dense network including thick (1-2 µm diameter) and thin (0.1 µm diameter) elastic fibers was seen in the ILM. Investigation of the ultrastructural organization of elastic fibers within the PBs will help to improve the knowledge of the AF elastic fiber interconnectivity within the ILM, PB and intra-lamellar regions.
F and G boxes represent magnified areas as shown in f and g images, respectively, to identify the orientation of elastic fibers in the PBs.
Quantitative analysis
SEM images were acquired from the sample surface, while the distance from the sample to the beam source was kept constant. Once elastic fibers were identified in the digested region, a low magnification was used as a starting point from which sequential images were acquired at the region of interest at progressively increasing magnifications.
Statistical analysis
Results
- Comparing control and digested samples
- Partition boundaries ultrastructure in the transverse plane (0°)
- Partition boundaries ultrastructure in the oblique plane (30°)
- Quantitative analysis
The elastic fibers within the PB formed a complex and dense network composed of thick (approximately 1–2 µm in diameter) and thin (approximately 0.1 µm in diameter) fibers ( Figure 3d–f ). The thick elastic fibers within the network were parallel to each other and made an angle with respect to the transverse plane, while connecting adjacent lamellae (Figure 3f). Ultrastructural organization of PB elastic fibers under different magnifications in the cross-sectional plane (0°).
Frequently occurring features of a network consisting of thin and thick elastic fibers across all nine digested samples (transverse plane (0°)) on 20 µm scale bar. Elastic fibers within the PB formed a complex network and appeared to connect collagen bundles in a CS lamellae. In Figures 5e-f, the orientation of collagen fibers from adjacent bundles is identified by white arrows, while they are interconnected by elastic fibers in the PB.
Shown are elastic fibers (indicated by white arrows) between two adjacent collagen bundles, whose cross-sections have been surrounded by white dashed lines and indicated by asterisks (e-f). Some elastic fibers bundle into adjacent collagen bundles and are oriented parallel to the collagen fibers (indicated by curved arrows). Whereas other elastic fibers appear to penetrate directly into adjacent bundles (indicated by straight arrows).
At the borders of the PB-adjacent collagen bundles, where elastic fibers appear to penetrate the adjacent bundles, two different forms of anchoring were observed. Some elastic fibers fused into the adjacent collagen bundles to become oriented parallel to the collagen fibers (indicated by curved arrows). While other elastic fibers appeared to penetrate directly into the adjacent bundles (indicated by straight arrows).
Similar to the cross-sectional plane, a dense network of thick and thin interconnecting elastic fibers (1.5 to 0.2 µm diameter, respectively) was observed as an important structural feature of PB in the oblique plane across all digested samples at 30° (Figures 6a-i). The quantitative analyzes (Figures 7a and 7c) show the directional coherence coefficient and the organization of elastic fibers in the PB of digested samples at two different cutting levels. The directional coherence coefficient and the orientation of elastic fibers were measured relative to the TCD plane (Figure 7b).
Directional coherence coefficient of elastic fibers in PBs reported as mean (95% CI) for all digested samples in two different section planes (a). There was no significant difference in directional coherence coefficient of elastic fibers between the two different cutting surfaces (p = 0.35).
Discussion
Like other SEM studies, identification of elastic fibers by their physical appearance and size is another limitation. Elastic fibers have been distinguished from other fibrous components as they are generally twisted or straight strands (0.2–1.5 µm in diameter) that sometimes branch to enter a course network [42–44] . Based on SEM images obtained from cross-sectional planes, the regular presence of PBs dividing the CS lamellae across the AF (Figures 2a, b, e, f and 3d, e) has a well-organized interconnecting structure consisting of elastic fibers, indicated.
Consistent with other studies, collagen bundles in the CS lamellae of AF provide a segmental structure that is surrounded by a network of elastic fibers in the PBs and the inter-lamellar matrix (Figure 3b) [33–35]. This division of AF by crossing PBs was seen in SEM images in the transverse cutting plane (Figure 2e–f). SEM imaging of a control sample did not reveal elastic fibers in the PBs due to the presence of matrix and collagen constituents, while PBs appear to form a constant or definite pattern that repeats at uniform intervals (indicated by stars in Figure 2a-b).
By removing the matrix and collagen fibers, it is now possible to visualize elastic fiber organization within the PBs, and to our knowledge this new interpretation of elastic fiber ultrastructure has not been previously reported. In both cutting planes (transverse and oblique), a dense network including thick (1–2 µm diameter) and thin (less than 0.2 µm diameter) elastic fibers was seen in the PBs (Figures 3f and 5f). Analysis of the ultrastructural organization of elastic fibers in the PBs in the oblique plane revealed that elastic fibers connect adjacent collagen bundles in a CS lamella by two mechanisms: fusing with and becoming oriented parallel to the collagen fibers; or by a sharp penetration into adjacent collagen bundles (figures 5g and j).
Based on the proposed 3D geometric model of PB (Figure 8), a possible function of elastic fibers in PB is to structurally reinforce adjacent collagen bundles during loading. Quantitative analysis revealed that there was no significant difference in the structural organization of the elastic fibers between the transverse and shear planes. While at least three principle orientations of elastic fibers in PB were identified, the relatively low magnitudes of the coherence coefficients would suggest that the organization of elastic fibers in PB represented a more isotropic than anisotropic structure.
Based on the presented results, one can speculate about the likely mechanical role of PBs in the AF. However, a study of the mechanical properties of the PBs is needed to provide new mechanical insights, and the current study represents the first steps towards this end. The same method that was carefully developed for the visualization of the elastic fiber organization in the ILM and lamellae was used to present an ultrastructural understanding of the elastic fiber network in the PBs, which is new knowledge not previously presented. Furthermore, with the new proposed 3D geometry of the PBs resulting from the current study (Figure 8), more accurate multiscale computer models of the disk can be developed, which will provide new insights into the mechanics of the disk.
Conclusion
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Guilford, The three-dimensional architecture of the elastic fiber network of the canine hepatic portal system, The American journal of anatomy. A detailed ultrastructural examination in the septal boundaries of the annulus fibrosus in the disc revealed a well-organized elastic fiber network with a complex ultrastructure. The density of the elastic fiber network in PBs was lower and the fiber orientation corresponded to the intra-lamellar space and the inter-lamellar matrix.
