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The stress relaxation of the TZ region, located at the interface of the Annulus Fibrosus (AF) and Nucleus Pulposus (NP) of the disc, and how its stress is relaxed compared to the adjacent regions is unknown. The current study aimed to identify the TZ stress relaxation properties under different strain magnitudes (0.2, 0.4, and 0.6 mm/mm) and compared the TZ stress relaxation characteristics to the NP and inner AF (IAF) regions at a specific strain magnitude (0.6 mm/mm). The results of the current study revealed that the TZ region exhibited different stress relaxation properties under various strain magnitudes with significantly higher initial (p < 0.008) and reduced stresses (marginally; p = 0.06) at higher strains. Our experimental stress relaxation data revealed a significantly higher equilibrium stress for the IAF compared to the TZ and NP regions (p < 0.001) but not between the TZ and NP regions (p = 0.7). We found that NP radial stress relaxed significantly faster (p < 0.04) than the TZ and NP. Additionally, the current study proposed a simple mathematical model and identified that, consistent with experimental data, the overall effect of region on both the level of decayed stress and the rate at which stress is relaxed was significant (p < 0.006). The current study found a similar stress relaxation characteristic between the NP and TZ regions, while IAF exhibited different stress relaxation properties. It is possible that this mismatch in stress relaxation acts as a shape transformation mechanism triggered by viscoelastic behavior. STATEMENT OF SIGNIFICANCE: Our understanding of the biomechanical properties of the transition zone (TZ) in the IVD, a region at the interface of the Nucleus Pulposus (NP) and Annulus Fibrosus (AF), is sparse. Unfortunately, there are no current studies that investigate the TZ stress relaxation properties and how stress is relaxed in the TZ compared to the adjacent regions. For the first time, the current study characterized the stress relaxation properties of the TZ and described how the TZ stress is relaxed compared to its adjacent regions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Understanding the structure-function relationship in the intervertebral disk (IVD) is crucial for the development of novel tissue engineering strategies to regenerate IVD and the establishment of accurate computational models for low back pain research. A large number of studies have improved our knowledge of the mechanical and structural properties of the nucleus pulposus (NP) and annulus fibrosus (AF), two of the main regions in the IVD. However, few studies have focused on the AF-NP interface (transition zone; TZ). Therefore, the current study aims to, for the first time, characterize the cyclic and failure mechanical properties of the TZ region under physiological loading (1, 3, and 5%s -1 strain rates) and investigate the structural integration mechanisms between the NP, TZ, and AF regions. The results of the current study reveal significant effects of region (NP, TZ, and AF) and strain rates (1, 3, and 5%s -1 ) on stiffness (p < 0.001). In addition, energy absorption is significantly higher for the AF compared to the TZ and NP (p <0.001) as well as between the TZ and NP (p <0.001). The current research finds adaptation, direct penetration, and entanglement between TZ and AF fibers as three common mechanisms for structural integration between the TZ and AF regions. STATEMENT OF SIGNIFICANCE: Despite a large number of studies that have mechanically, structurally, and biologically characterized the nucleus pulposus (NP) and annulus fibrosus (AF) regions, few studies have focused on the NP-AF interface region (known as Transition Zone; TZ) in the IVD; hence, our understanding of the TZ structure-function relationship is still incomplete. Of particular importance, the cyclic mechanical properties of the TZ, compared to the adjacent regions (NP and AF), are yet to be explored and the precise nature of the structural integration between the NP and AF via the TZ region is not yet known. The current study explores both the mechanical and structural properties of the TZ region to ultimately identify the mechanism of integration between the NP and AF., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Despite extensive efforts over the past 40 years, there is still a significant gap in knowledge of the characteristics of elastic fibers in the intervertebral disc (IVD). More studies are required to clarify the potential contribution of elastic fibers to the IVD (healthy and diseased) function and recommend critical areas for future investigations. On the other hand, current IVD in-vitro models are not true reflections of the complex biological IVD tissue and the role of elastic fibers has often been ignored in developing relevant tissue-engineered scaffolds and realistic computational models. This has affected the progress of IVD studies (tissue engineering solutions, biomechanics, fundamental biology) and translation into clinical practice. Motivated by the current gap, the current review paper presents a comprehensive study (from the early 1980s to 2022) that explores the current understanding of structural (multi-scale hierarchy), biological (development and aging, elastin content, and cell-fiber interaction), and biomechanical properties of the IVD elastic fibers, and provides new insights into future investigations in this domain.

The Nucleus Pulposus (NP) and Annulus Fibrous (AF) are two primary regions of the intervertebral disc (IVD). The interface between the AF and NP, where the gradual transition in structure and type of fibers are observed, is known as the Transition Zone (TZ). Recent structural studies have shown that the TZ contains organized fibers that appear to connect the NP to the AF. However, the mechanical characteristics of the TZ are yet to be explored. The current study aimed to investigate the mechanical properties of the TZ at the anterolateral (AL) and posterolateral (PL) regions in both radial and circumferential directions of loading using ovine IVDs (N = 28). Young's and toe moduli, maximum stress, failure strain, strain at maximum stress, and toughness were calculated mechanical parameters. The findings from this study revealed that the mechanical properties of the TZ, including young's modulus (p = 0.001), failure strain (p < 0.001), strain at maximum stress (p = 0.002), toughness (p = 0.027), and toe modulus (p = 0.005), were significantly lower for the PL compared to the AL region. Maximum stress was not significantly different between the PL and AL regions (p = 0.164). We found that maximum stress (p = 0.002), failure strain (p < 0.001), and toughness (p = 0.001) were significantly different in different loading directions. No significant differences for modulus (young's; p = 0.169 and toe; p = 0.352) and strain at maximum stress (p = 0.727) were found between the radial and circumferential loading directions. STATEMENT OF SIGNIFICANCE: To date there has not been a study that has investigated the mechanical characterization of the annulus (AF)-nucleus (NP) interface (transition zone; TZ) in the intervertebral disc (IVD), nor is it known whether the posterolateral (PL) and anterolateral (AL) regions of the TZ exhibit different mechanical properties. Accordingly, the TZ mechanical properties have been rarely used in the development of computational IVD models and relevant tissue-engineered scaffolds. The current research reported the mechanical properties of the TZ region and revealed that its mechanical properties were significantly lower for the PL compared to the AL region. These new findings enhance our knowledge about the nature of AF-NP integration and may help to develop more realistic tissue-engineered or computational IVD models., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Collagen fibers within the annulus fibrosus (AF) lamellae are unidirectionally aligned with alternating orientations between adjacent layers. AF constitutive models often combine two adjacent lamellae into a single equivalent layer containing two fiber networks with a crisscross pattern. Additionally, AF models overlook the inter-lamellar matrix (ILM) as well as elastic fiber networks in between lamellae. We developed a nonhomogenous micromechanical model as well as two coarser homogenous hyperelastic and microplane models of the human AF, and compared their performances against measurements (tissue level uniaxial and biaxial tests as well as whole disc experiments) and seven published hyperelastic models. The micromechanical model had a realistic non-homogenous distribution of collagen fiber networks within each lamella and elastic fiber network in the ILM. For small matrix linear moduli (<0.2 MPa), the ILM showed substantial anisotropy (>10%) due to the elastic fiber network. However, at moduli >0.2 MPa, the effects of the elastic fiber network on differences in stress-strain responses at different directions disappeared (<10%). Variations in sample geometry and boundary conditions (due to uncertainty) markedly affected stress-strain responses of the tissue in uniaxial and biaxial tests (up to 16 times). In tissue level tests, therefore, simulations should represent testing conditions (e.g., boundary conditions, specimen geometry, preloads) as closely as possible. Stress/strain fields estimated from the single equivalent layer approach (conventional method) yielded different results from those predicted by the anatomically more accurate apparoach (i.e., layerwise). In addition, in a disc under a compressive force (symmetric loading), asymmetric stress-strain distributions were computed when using a layerwise simulation. Although all developed and selected published AF models predicted gross compression-displacement responses of the whole disc within the range of measured data, some showed excessively stiff or compliant responses under tissue-level uniaxial/biaxial tests. This study emphasizes, when constructing and validating constitutive models of AF, the importance of the proper simulation of individual lamellae as distinct layers, and testing parameters (sample geometric dimensions/loading/boundary conditions)., Competing Interests: Declaration of Competing Interest None, (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

There has been no study to describe the ultrastructural organization of elastic fibers at the interface of the nucleus pulposus and annulus fibrosus of the intervertebral disk (IVD), a region called the transition zone (TZ). A previously developed digestion technique was optimized to eliminate cells and non-elastin ECM components except for the elastic fibers from the anterolateral (AL) and posterolateral (PL) regions of the TZ in ovine IVDs. Not previously reported, the current study identified a complex elastic fiber network across the TZ for both AL and PL regions. In the AL region, this network consisted of major thick elastic fibers (≈ 1 µm) that were interconnected with delicate (< 200 nm) elastic fibers. While the same ultrastructural organization was observed in the PL region, interestingly the size of the elastic fibers was smaller (< 100 nm) compared to those that were located in the AL region. Quantitative analysis of the elastic fibers revealed significant differences in the size (p < 0.001) and the orientation of elastic fibers (p = 0.001) between the AL and PL regions, with a higher orientation and larger size of elastic fibers observed in the AL region. The gradual elimination of cells and non-elastin extracellular matrix components identified that elastic fibers in the TZ region in combination with the extracellular matrix created a honeycomb structure that was more compact at the AF interface compared to that located close to the NP. Three different symmetrically organized angles of rotation (0⁰ and ±90⁰) were detected for the honeycomb structure at both interfaces, and the structure was significantly orientated at the TZ-AF compared to the TZ-NP interface (p = 0.003)., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier Ltd.)

Damage to the annulus fibrosus (AF), the outer region of the intervertebral disc (IVD), results in an undesirable condition that may accelerate IVD degeneration causing low back pain. Despite intense research interest, attempts to regenerate the IVD have failed so far and no effective strategy has translated into a successful clinical outcome. Of particular significance, the failure of strategies to repair the AF has been a major drawback in the regeneration of IVD and nucleus replacement. It is unlikely to secure regenerative mediators (cells, genes, and biomolecules) and artificial nucleus materials after injection with an unsealed AF, as IVD is exposed to significant load and large deformation during daily activities. The AF defects strongly change the mechanical properties of the IVD and activate catabolic routes that are responsible for accelerating IVD degeneration. Therefore, there is a strong need to develop effective therapeutic strategies to prevent or reconstruct AF damage to support operational IVD regenerative strategies and nucleus replacement. By the way of this review, repair and regenerative strategies for AF reconstruction, their current status, challenges ahead, and future outlooks were discussed.

Study Design: A biomechanical analysis correlating internal disc strains and tissue damage during simulated repetitive lifting., Objective: To understand the failure modes during simulated safe and unsafe repetitive lifting., Summary of Background Data: Repetitive lifting has been shown to lead to lumbar disc herniation (LDH). In vitro studies have developed a qualitative understanding of the effect of repetitive loading on LDH. However, no studies have measured internal disc strains and subsequently correlated these with disc damage., Methods: Thirty human cadaver lumbar functional spinal units were subjected to an equivalent of 1 year of simulated repetitive lifting under safe and unsafe levels of compression, in combination with flexion (13-15°), and right axial rotation (2°) for 20,000 cycles or until failure. Safe or unsafe lifting were applied as a compressive load to mimic holding a 20 kg weight either close to, or at arm's length, from the body, respectively. Maximum shear strains (MSS) were measured, and disc damage scores were determined in nine regions from axial post-test magnetic resonance imaging (MRI) and macroscopic images., Results: Twenty percent of specimens in the safe lifting group failed before 20,000 cycles due to endplate failure, compared with 67% in the unsafe group. Over half of the specimens in the safe lifting group failed via either disc protrusion or LDH, compared with only 20% via protrusion in the unsafe group. Significant positive correlations were found between MRI and macroscopic damage scores in all regions (rs > 0.385, P < 0.049). A significant positive correlation was observed in the left lateral region for MSS versus macroscopic damage score (rs = 0.486, P < 0.037) and MSS versus failure mode (rs = 0.724, P = 0.018, only specimens with disc failure). Pfirrmann Grade 3 discs were strongly associated with subsequent LDH (P = 0.003)., Conclusion: Increased shear strains were observed in the contralateral side to the applied rotation as disc injury progressed from protrusion to LDH. Larger compressive loads applied to simulate unsafe lifting led to frequent early failure of the endplate, however, smaller compressive loads at similar flexion angles applied under safe lifting led to more loading cycles before failure, where the site of failure was more likely to be the disc. Our study demonstrated that unsafe lifting leads to greater risk of injury compared with safe lifting, and LDH and disc protrusion were more common in the posterior/posterolateral regions., Level of Evidence: N/A.

The inter-lamellar matrix (ILM)-located between adjacent lamellae of the annulus fibrosus-consists of a complex structure of elastic fibers, while elastic fibers of the intra-lamellar region are aligned predominantly parallel to the collagen fibers. The organization of elastic fibers under low magnification, in both inter- and intra-lamellar regions, was studied by light microscopic analysis of histologically prepared samples; however, little is known about their ultrastructure. An ultrastructural visualization of elastic fibers in the inter-lamellar matrix is crucial for describing their contribution to structural integrity, as well as mechanical properties of the annulus fibrosus. The aims of this study were twofold: first, to present an ultrastructural analysis of the elastic fiber network in the ILM and intra-lamellar region, including cross section (CS) and in-plane (IP) lamellae, of the AF using Scanning Electron Microscopy (SEM) and second, to -compare the elastic fiber orientation between the ILM and intra-lamellar region. Four samples (lumbar sheep discs) from adjacent sections (30μm thickness) of anterior annulus were partially digested by a developed NaOH-sonication method for visualization of elastic fibers by SEM. Elastic fiber orientation and distribution were quantified relative to the tangential to circumferential reference axis. Visualization of the ILM under high magnification revealed a dense network of elastic fibers that has not been previously described. Within the ILM, elastic fibers form a complex network, consisting of different size and shape fibers, which differed to those located in the intra-lamellar region. For both regions, the majority of fibers were oriented near 0° with respect to tangential to circumferential (TCD) direction and two minor symmetrical orientations of approximately±45°. Statistically, the orientation of elastic fibers between the ILM and intra-lamellar region was not different (p=0.171). The present study used extracellular matrix partial digestion to address significant gaps in understanding of disc microstructure and will contribute to multidisciplinary ultrastructure-function studies., Statement of Significance: Visualization of the intra-lamellar matrix under high magnification revealed a dense network of elastic fibers that has not been previously described. The present study used extracellular matrix partial digestion to address significant gaps in understanding of disc microstructure and will contribute to multidisciplinary ultrastructure-function studies., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Collagen and elastic fibers are two major fibrous constituents of the annulus fibrosus (AF) in the disc that contribute to its mechanical and viscoelastic properties. It was thought that elastic fibers play no substantial role in the function and properties of the disc as these fibers were irregularly distributed. Studies that have revealed highly organized elastic fibers with different regional orientation and distribution, while being strongly crosslinked with matrix, suggesting their contribution to disc structure-function properties. These studies that were performed by light microscopic analysis of histologically prepared samples, have not been able to reveal the fine-scale architectural details of the elastic fiber network. Since elastic fibers are intermingled with other fibrous components of the disc and mostly obscured by the extracellular matrix, it is difficult to demonstrate their ultra-structural organization using scanning electron microscopy (SEM). Therefore the aim of this study was to develop a rapid matrix digestion technique for ultrastructural analysis of the disc elastic fibers. This study provides a new method for fundamental visualization of elastic fibers and their architecture in the disc. Through the ultra-structural analysis, the relationship between structure and function, as well as the role of elastic fibers on AF mechanical properties can be studied. This method may be used to develop a three-dimensional map of elastic fibers distribution within the disc, which would provide valuable information for designing tissue engineered scaffolds for AF repair and replacement., (Copyright © 2017 Elsevier Ltd. All rights reserved.)