Elastic fibers: The missing key to improve engineering concepts for reconstruction of the Nucleus Pulposus in the intervertebral disc.

The increasing prevalence of low back pain has imposed a heavy economic burden on global healthcare systems. Intense research activities have been performed for the regeneration of the Nucleus Pulposus (NP) of the IVD; however, tissue-engineered scaffolds have failed to capture the multi-scale structural hierarchy of the native tissue. The current study revealed for the first time, that elastic fibers form a network across the NP consisting of straight and thick parallel fibers that were interconnected by wavy fine fibers and strands. Both straight fibers and twisted strands were regularly merged or branched to form a fine elastic network across the NP. As a key structural feature, ultrathin (53 ± 7 nm), thin (215 ± 20 nm), and thick (890 ± 12 nm) elastic fibers were observed in the NP. While our quantitative analysis for measurement of the thickness of elastic fibers revealed no significant differences (p < 0.633), the preferential orientation of fibers was found to be significantly different (p < 0.001) across the NP. The distribution of orientation for the elastic fibers in the NP represented one major organized angle of orientation except for the central NP. We found that the distribution of elastic fibers in the central NP was different from those located in the peripheral regions representing two symmetrically organized major peaks (±45⁰). No significant differences in the maximum fiber count at the major angles of orientation (±45⁰) were observed for both peripheral (p = 0.427) and central NP (p = 0.788). Based on these new findings a structural model for the elastic fibers in the NP was proposed. The geometrical presentation, along with the distribution of elastic fibers orientation, resulting from the present study identifies the ultrastructural organization of elastic fibers in the NP important towards understanding their mechanical role which is still under investigation. Given the results of this new geometrical analysis, more-accurate multiscale finite element models can now be developed, which will provide new insights into the mechanobiology of the IVD. In addition, the results of this study can potentially be used for the fabrication of bio-inspired tissue-engineered scaffolds and IVD models to truly capture the multi-scale structural hierarchy of IVDs. STATEMENT OF SIGNIFICANCE: Visualization of elastic fibers in the nucleus of the intervertebral disk under high magnification was not reported before. The present research utilized extracellular matrix partial digestion to address significant gaps in understanding of nucleus microstructure that can potentially be used for the fabrication of bio-inspired tissue-engineered scaffolds and disk models to truly capture the multi-scale structural hierarchy of discs.