Annulus Fibrosus Regions Research Articles

3D bioprinting of an intervertebral disc tissue analogue with a highly aligned annulus fibrosus via suspended layer additive manufacture.

Intervertebral disc (IVD) function is achieved through integration of its two component regions: the nucleus pulposus (NP) and the annulus fibrosus (AF). The NP is soft (0.3-5 kPa), gelatinous and populated by spherical NP cells in a polysaccharide-rich extracellular matrix (ECM). The AF is much stiffer (~100 kPa) and contains layers of elongated AF cells in an aligned, fibrous ECM. Degeneration of the disc is a common problem with age being a major risk factor. Progression of IVD degeneration leads to chronic pain and can result in permanent disability. The development of therapeutic solutions for IVD degeneration is impaired by a lack of in vitro models of the disc that are capable of replicating the fundamental structure and biology of the tissue. This study aims to investigate if a newly developed suspended hydrogel bioprinting system (termed SLAM) could be employed to fabricate IVD analogues with integrated structural and compositional features similar to native tissue. Bioprinted IVD analogues were fabricated to recapitulate structural, morphological and biological components present in the native tissue. The constructs replicated key structural components of native tissue with the presence of a central, polysaccharide-rich NP surrounded by organised, aligned collagen fibres in the AF. Cell tracking, actin and matrix staining demonstrated that embedded NP and AF cells exhibited morphologies and phenotypes analogous to what is observed in vivo with elongated, aligned AF cells and spherical NP cells that deposited HA into the surrounding environment. Critically, it was also observed that the NP and AF regions contained a defined cellular and material interface and segregated regions of the two cell types, thus mimicking the highly regulated structure of the IVD.

Innovative hydrogel-patch combination for large annulus fibrosus defects: a prospective approach to address herniation recurrence

BACKGROUND CONTEXTLarge annulus fibrosus (AF) defects often lead to a high rate of reherniation, particularly in the medial AF region, which has limited self-healing capabilities. The increasing prevalence of herniated discs underscores the need for effective repair strategies. PURPOSEThe objectives of this study were to design an AF repair technique to reduce solve the current problems of insufficient mechanical properties and poor sealing capacity. STUDY DESIGNIn vitro biomechanical experiments and finite element analysis. METHODSThe materials used in this study were patches and hydrogels with good biocompatibility and sufficient mechanical properties to withstand loading in the lumbar spine. Five repair techniques were assessed in this study: hydrogel filler (HF), AF patch medial barrier (MB), AF patch medial barrier and hydrogel filler (MB&HF), AF patch medial-lateral barrier (MLB), and AF patch medial-lateral barrier and hydrogel filler (MLB&HF). The repair techniques were subjected to in vitro testing (400 N axial compression and 0–500 N fatigue loading at 5Hz) and finite element analysis (400 N axial compression) to evaluate the effectiveness at repairing large AF defects. The evaluation included repair tightness, spinal stability, and fatigue resistance. RESULTSFrom the in vitro testing, the failure load of the repair techniques was in the following order HF <MB <MB&HF <MLB <MLB&HF. Both HF and MB groups failed to effectively increase intervertebral disc (IVD) stiffness, resulting in a reduction in spatial stability. The MLB, MB&HF, and MLB&HF groups partially restored IVD stiffness, with MLB&HF showing the most effective recovery (−24.13% ± 3.59%). From the finite element models, incorporating a hydrogel filler was best able to maintain the IVD height. Patch repair alone could not adequately reduce the high AF stress due to AF injury, but with hydrogel support, stress was substantially low and more uniformly distributed. All repair techniques demonstrated reduced stress around the damaged area on the AF, in comparison to the unrepaired model. The NP pressure in the HF group was closest to the intact group, and the patch repair reduced the NP pressure. The maximum patch deformation and suture stress were ranked as MB >MLB >MB&HF >MLB&HF. CONCLUSIONSThe combined use of patches and hydrogels exhibited promising mechanical properties postdiscectomy, providing a promising solution for addressing large AF defects and improving disc stability. CLINICAL SIGNIFICANCEThis study introduces a promising method for repairing large annular fissure (AF) defects after disc herniation, combining patch repair with a hydrogel filler. These techniques hold potential for developing clinical AF repair products to address this challenging issue.

Injectable Radiopaque Hyaluronic Acid Granular Hydrogels for Intervertebral Disc Repair.

Injectable hydrogels offer minimally-invasive treatment options for degenerative disc disease, a prevalent condition affecting millions annually. Many hydrogels explored for intervertebral disc (IVD) repair suffer from weak mechanical integrity, migration issues, and expulsion. To overcome these limitations, an injectable and radiopaque hyaluronic acid granular hydrogel is developed. The granular structure provides easy injectability and low extrusion forces, while the radiopacity enables direct visualization during injection into the disc and non-invasive monitoring after injection. The radiopaque granular hydrogel is injected into rabbit disc explants to investigate restoration of healthy disc mechanics following needle puncture injury ex vivo and then delivered in a minimally-invasive manner into the intradiscal space in a clinically-relevant in vivo large animal goat model of IVD degeneration initiated through degradation by chondroitinase. The radiopaque granular hydrogel successfully halted loss of disc height due to degeneration. Further, the hydrogel not only enhanced proteoglycan content and reduced collagen content in the nucleus pulposus (NP) region compared to degenerative discs, but also helped to maintain the structural integrity of the disc and promote healthy segregation of the NP and annulus fibrosus regions. Overall, this study demonstrates the great potential of an injectable radiopaque granular hydrogel for treatment of degenerative disc disease.

Structure-function characterization of the transition zone in the intervertebral disc.

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.

Epigenetic Changes within the Annulus Fibrosus by DNA Methylation in Rat Intervertebral Disc Degeneration Model.

Intervertebral disc degeneration (IDD) is an age-dependent progressive spinal disease that causes chronic back or neck pain. Although aging has long been presented as the main risk factor, the exact cause is not fully known. DNA methylation is associated with chronic pain, suggesting that epigenetic modulation may ameliorate disc degeneration. We examined histological changes in the DNA methylation within the discs and their association with pain-related transient receptor potential vanilloid subtype 1 (TrpV1) expression in rats subjected to IDD. Epigenetic markers (5-hydroxymethylcytosine (5hmC), 5-methylcytosine (5Mc)), DNA methyltransferases (DNMTs), and Ten-eleven translocations (Tets) were analyzed using immunohistochemistry, real-time PCR, and DNA dot-blot following IDD. Results revealed high 5mC levels in the annulus fibrosus (AF) region within the disc after IDD and an association with TrpV1 expression. DNMT1 is mainly involved in 5mC conversion in degenerated discs. However, 5hmC levels did not differ between groups. A degenerated disc can lead to locomotor defects as assessed by ladder and tail suspension tests, no pain signals in the von Frey test, upregulated matrix metalloproteinase-3, and downregulated aggrecan levels within the disc. Thus, we found that the DNA methylation status in the AF region of the disc was mainly changed after IDD and associated with aberrant TrpV1 expression in degenerated discs.

Biomechanical effects of cement discoplasty on the lumbar spinal unit.

Percutaneous cement discoplasty (PCD) is used to treat patients with low back and leg pain due to the intervertebral disc vacuum phenomena. Whether PCD can restore lumbar spinal stability remains unknown. The purpose of our in vitro study was to evaluate the biomechanical changes brought about by PCD. Eight fresh pig lumbar spines were tested in the following order: intact, after nucleotomy, and after discoplasty. Flexion/extension, lateral bending, and axial rotation were induced by pure moments. The range of motion and neutral zone were recorded. A CT scan was performed to assess the injection volume of the bone cement and to observe whether the bone cement was fractured. After removing the facet joint, a compression failure test was conducted to observe the fracture of bone cement. Compared with nucleotomy, range of motion (ROM) after discoplasty was reduced only in lateral flexion (P < 0.05). The results of the neutral zone showed that the neutral zones in flexion-extension and lateral bending were significantly reduced after discoplasty (P < 0.05). The neutral zone was more sensitive to changes in lumbar stability than ROM. Bone cement slides were observed during the biomechanical test. The CT scan and compression failure test showed that bone cement fracture was more likely to occur at the puncture channel in the annulus fibrosus region. In all, the biomechanical study indicates that discoplasty helps enhance the stability of the lumbar spine in flexion-extension and lateral bending, which explains how PCD works for low back pain. Fractures and sliding of bone cement were observed after discoplasty, and this was more likely to occur at the puncture channel in the annulus fibrosus region. This suggests that bone cement displacement after PCD may cause nerve compression.

Magnetic Resonance Elastography of Intervertebral Discs: Spin-Echo Echo-Planar Imaging Sequence Validation.

Magnetic resonance elastography (MRE) is an imaging technique that can noninvasively assess the shear properties of the intervertebral disc (IVD). Unlike the standard gradient recalled echo (GRE) MRE technique, a spin-echo echo-planar imaging (SE-EPI) sequence has the potential to improve imaging efficiency and patient compliance. To validate the use of an SE-EPI sequence for MRE of the IVD compared against the standard GRE sequence. Cross-over. Twenty-eight healthy volunteers (15 males and 13 females, age range: 19-55). 3 T; GRE, SE-EPI with breath holds (SE-EPI-BH) and SE-EPI with free breathing (SE-EPI-FB) MRE sequences. MRE-derived shear stiffnesses were calculated via principal frequency analysis. SE-EPI derived shear stiffness and octahedral shear strain signal-to-noise ratios (OSS-SNR) were compared against those derived using the GRE sequence. The reproducibility and repeatability of SE-EPI stiffness measurements were determined. Shear stiffness was evaluated in the nucleus pulposus (NP) and annulus fibrosus (AF) regions of the disc. Scan times between sequences were compared. Linear mixed models, Bland-Altman plots, and Lin's concordance correlation coefficients (CCCs) were used with P < 0.05 considered statistically significant. Good correlation was observed between shear stiffnesses derived from the SE-EPI sequences with those derived from the GRE sequence with CCC values greater than 0.73 and 0.78 for the NP and AF regions, respectively. OSS-SNR was not significantly different between GRE and SE-EPI sequences (P > 0.05). SE-EPI sequences generated highly reproducible and repeatable stiffness measurements with CCC values greater than 0.97 in the NP and AF regions and reduced scan time by at least 51% compared to GRE. SE-EPI-BH and SE-EPI-FB stiffness measurements were similar with CCC values greater than 0.98 for both regions. SE-EPI-based MRE-derived stiffnesses were highly reproducible and repeatable and correlated with current standard GRE MRE-derived stiffness estimates while reducing scan times. 1 TECHNICAL EFFICACY STAGE: 1.

Preclinical ex-vivo Testing of Anti-inflammatory Drugs in a Bovine Intervertebral Degenerative Disc Model.

Discogenic low back pain (LBP) is a main cause of disability and inflammation is presumed to be a major driver of symptomatic intervertebral disc degeneration (IDD). Anti-inflammatory agents are currently under investigation as they demonstrated to alleviate symptoms in patients having IDD. However, their underlying anti-inflammatory and regenerative activity is poorly explored. The present study sought to investigate the potential of Etanercept and Tofacitinib for maintaining disc homeostasis in a preclinical intervertebral disc (IVD) organ culture model within IVD bioreactors allowing for dynamic loading and nutrient exchange. Bovine caudal IVDs were cultured in a bioreactor system for 4 days to simulate physiological or degenerative conditions: (1) Phy—physiological loading (0.02–0.2 MPa; 0.2 Hz; 2 h/day) and high glucose DMEM medium (4.5 g/L); (2) Deg+Tumor necrosis factor α (TNF-α)—degenerative loading (0.32–0.5 MPa; 5 Hz; 2 h/day) and low glucose DMEM medium (2 g/L), with TNF-α injection. Etanercept was injected intradiscally while Tofacitinib was supplemented into the culture medium. Gene expression in the IVD tissue was measured by RT-qPCR. Release of nitric oxide (NO), interleukin 8 (IL-8) and glycosaminoglycan (GAG) into the IVD conditioned medium were analyzed. Cell viability in the IVD was assessed using lactate dehydrogenase and ethidium homodimer-1 staining. Immunohistochemistry was performed to assess protein expression of IL-1β, IL-6, IL-8, and collagen type II in the IVD tissue. Etanercept and Tofacitinib downregulated the expression of IL-1β, IL-6, IL-8, Matrix metalloproteinase 1 (MMP1), and MMP3 in the nucleus pulposus (NP) tissue and IL-1β, MMP3, Cyclooxygenase-2 (COX2), and Nerve growth factor (NGF) in the annulus fibrosus (AF) tissue. Furthermore, Etanercept significantly reduced the IL-1β positively stained cells in the outer AF and NP regions. Tofacitinib significantly reduced IL-1β and IL-8 positively stained cells in the inner AF region. Both, Etanercept and Tofacitinib reduced the GAG loss to the level under physiological culture condition. Etanercept and Tofacitinib are able to neutralize the proinflammatory and catabolic environment in the IDD organ culture model. However, combined anti-inflammatory and anabolic treatment may be required to constrain accelerated IDD and relieving inflammation-induced back pain.

Engineering a biomimetic integrated scaffold for intervertebral disc replacement

Tissue engineering technology provides a promising alternative to restore physiological functionality of damaged intervertebral disc (IVD). Advanced tissue engineering strategies for IVD have increasingly focused on engineering IVD regions combined the inner nucleus pulposus (NP) and surrounding annulus fibrosus (AF) tissue. However, simulating the cellular and matrix structures and function of the complex structure of IVD is still a critical challenge. Toward this goal, this study engineered a biomimetic AF-NP composite with circumferentially oriented poly(ε-caprolactone) microfibers seeded with AF cells, with an alginate hydrogel encapsulating NP cells as a core. Fluorescent imaging and histological analysis showed that AF cells spread along the circumferentially oriented PCL microfibers and NP cells colonized in the alginate hydrogel similar to native IVD, without obvious migration and mixing between the AF and NP region. Engineered IVD implants showed progressive tissue formation over time after subcutaneous implantation in nude mice, which were indicated by deposition and organization of extracellular matrix and enhanced mechanical properties. In terms of form and function of IVD-like tissue, our engineered biomimetic AF-NP composites have potential application for IVD replacement.

Decellularization and characterization of a whole intervertebral disk xenograft scaffold.

Intervertebral disk (IVD) degeneration is a multifactor process that results in the physical destruction of the nucleus pulposus (NP) and annulus fibrosus (AF). This compromises IVD function and causes significant disability and economic burden. Strategies to replace the entire composite structure of the IVD are limited and most approaches do not recapitulate the heterogenous biochemical composition, microarchitecture or mechanical properties of the native tissue. Our central hypothesis was that donor IVDs which resemble the size and biochemistry of human lumbar IVDs could be successfully decellularized while retaining the tissue's structure and function with the long-term goal of creating a composite scaffold for tissue engineering the human IVD. Accordingly, we optimized a procedure to decellularize bovine tail IVDs using a combination of detergents, ultrasonication, freeze-thaw cycles, and nucleases. The resultant decellularized whole IVD xenografts retained distinct AF and NP regions which contained no visible intact cell nuclei and minimal residual bovine deoxyribose nucleic acid (DNA; 65.98 ± 4.07 and 47.12 ± 13.22 ng/mg, respectively). Moreover, the NP region of decellularized IVDs contained 313.40 ± 50.67 µg/mg glycosaminoglycan. The presence of collagen type II was confirmed via immunohistochemistry. Additionally, histological analysis of the AF region of decellularized IVDs demonstrated retention of the native angle-ply collagen microarchitecture. Unconfined compression testing demonstrated no significant differences in swelling pressure and toe-region modulus between fresh and decellularized IVDs. However, linear region moduli, peak stress and equilibrium moduli were all significantly reduced. Together, this research demonstrates a successful initial step in developing a biomimetic acellular whole IVD xenograft scaffold for use in IVD tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2412-2423, 2018.

Comparison of Oxygen Consumption Rates of Nondegenerate and Degenerate Human Intervertebral Disc Cells.

In vitro measurements of the oxygen consumption rates (OCR) of human intervertebral disc (IVD) cells. The aim of this study was to determine the differences in the OCR of nondegenerate and degenerate human annulus fibrosus (AF), nucleus pulposus (NP), and cartilage endplate (CEP) cells at different glucose concentrations. The avascular nature of the IVD creates a delicate balance between rate of nutrient transport through the matrix and rate of disc cell consumption necessary to maintain tissue health. Previous studies have shown a dependence of OCR for animal (e.g., bovine and porcine) IVD cells on oxygen level and glucose concentration. However, the OCR of nondegenerate human IVD cells compared to degenerate human IVD cells at different glucose concentrations has not been investigated. IVD cells were isolated from the AF, NP, and CEP regions of human cadaver spines and surgical samples. The changes in oxygen concentration were recorded when cells were sealed in a metabolic chamber. The OCR of cells was determined by curve fitting using the Michaelis-Menton equation. Under identical cell culture conditions, the OCR of degenerate human IVD cells was three to five times greater than that of nondegenerate human IVD cells. The degenerate IVD cells cultured in low-glucose medium (1 mmol/L) exhibited the highest OCR compared to degenerate cells cultured at higher glucose levels (i.e., 5 mmol/L, 25 mmol/L), whereas no significant differences in OCR were found among the nondegenerate IVD cells for all glucose levels. Considering the significantly higher OCR and unique response to glucose of degenerate human IVD cells, the degeneration of the IVD is associated with a cell phenotypic change related to OCR. The OCR of IVD cells reported in this study will be valuable for understanding human IVD cellular behavior and tissue nutrition in response to disc degeneration. N/A.

Diffusion-weighted 7.0T Magnetic Resonance Imaging in Assessment of Intervertebral Disc Degeneration in Rats.

Background:Intervertebral disc degeneration (IDD) is a major cause of disc protrusion, likely to be associated with decrease of water content. This research aimed to evaluate IDD by diffusion-weighted imaging (DWI) with a 7.0 Tesla (T) magnetic resonance imaging (MRI) machine.Methods:A total of 24 healthy Sprague-Dawley rats were randomly selected and divided into four groups (A, B, C, and D), each consisting of 3 male and 3 female rats (28, 42, 56, and 70 days old, respectively). All the rats were imaged with a 7.0T MRI, producing T2WI, T1WI, and functional DWI sequences. Data were collected and apparent diffusion coefficient (ADC) charts were constructed. Nucleus pulposus and annulus fibrosus regions were identified, several regions of interest were chosen, and their ADC values were obtained. After imaging, rats were sacrificed and their intervertebral discs (L1–L6) were dissected, yielding a total of 144 discs. Protein was extracted for the purpose of Western blotting. Comparison among multiple samples used one-way analysis of variance and least significant difference methods.Results:7.0T MRI revealed evident decrease in signal intensity within intervertebral discs of Sprague-Dawley rats with age. Intervertebral disc ADC values significantly decreased from Group A (0.00154 ± 0.00008) to Group D (0.00107 ± 0.00007; P < 0.01); nucleus pulposus ADC values significantly decreased from Group A (0.00164 ± 0.00005) to Group D (0.00140 ± 0.00007; P < 0.01) and annulus fibrosus ADC values significantly decreased from Group A (0.00129 ± 0.00014) to Group D (0.00082 ± 0.00012; P < 0.01). Meanwhile, it also revealed evident decrease from high spinal level to low spinal level: nucleus pulposus ADC values in Group A significantly decreased from L1/L2 (0.00163 ± 0.00006) to L6/S1 (0.00139 ± 0.00004; P < 0.01). While annulus fibrosus ADC values did not differ significantly between levels in Group A (P > 0.05). Western blotting showed that aggrecan content of intervertebral discs decreased from Group A (1.88 ± 0.16) to Group D (0.17 ± 0.04) with age (P < 0.01); Type II collagen content of intervertebral discs decreased from Group A (2.22 ± 0.04) to Group D (0.20 ± 0.01) with age (P < 0.01). No significant differences in aggrecan and Type II collagen content of L1–L6 intervertebral discs in Group A were noted (P > 0.05). Mean ADC values of different intervertebral regions were positively correlated with aggrecan and Type II collagen content (aggrecan: r = 0.631, P < 0.01; Type II collagen: r = 0.680, P < 0.01).Conclusion:7.0T MRI-DWI could be applied to effectively diagnose and research early IDD in tiny variations.

Transplantation of Hypoxic-Preconditioned Bone Mesenchymal Stem Cells Retards Intervertebral Disc Degeneration via Enhancing Implanted Cell Survival and Migration in Rats.

Objective Special hypoxic and hypertonic microenvironment in intervertebral discs (IVDs) decreases the treatment effect of cell transplantation. We investigated the hypothesis that hypoxic preconditioning (HP) could improve the therapeutic effect of bone mesenchymal stem cells (BMSCs) to IVD degeneration. Methods BMSCs from green fluorescent protein-transgenic rats were pretreated with cobalt chloride (CoCl2, 100 μM, 24 h) for hypoxic conditions in vitro. Apoptosis (related pathways of caspase-3 and bcl-2) and migration (related pathways of HIF-1α and CXCR4) were detected in BMSCs. In vivo, BMSCs and HP BMSCs (H-BMSCs) were injected into the rat model of IVD degeneration. The IVD height, survival, migration, and differentiation of transplanted BMSCs and matrix protein expression (collagen II, aggrecan, and MMP-13) were tested. Results H-BMSCs could extensively decrease IVD degeneration by increasing IVD height and collagen II and aggrecan expressions when compared with BMSCs. Significantly, more GFP-positive BMSCs were observed in the nucleus pulposus and annulus fibrosus regions of IVD. HP could significantly decrease BMSC apoptosis (activating bcl-2 and inhibiting caspase-3) and improve BMSC migration (increasing HIF-1α and CXCR4) in vitro. Conclusion HP could significantly enhance the capacity of BMSCs to repair DDD by increasing the survival and migration of implanted cells and increasing matrix protein expression.

An anisotropic multiphysics damage model with application to annulus fibrosus

An anisotropic multiphysics damage model is developed to characterize the couplings among multiple physical fields within soft tissues and the tissue damage based on thermodynamic principles. This anisotropic multiphysics damage model integrates the continuum mixture theory and a continuum damage model, and the anisotropic damage is considered by evolution of internal damage variables governing the anisotropic mechanical behaviors of tissues. The energy dissipation associated with the transport of fluid and ions is generally related to tissue damage. The anisotropic multiphysics damage model is applied to simulate a case of annulus fibrosus (AF) damage in an isolated intervertebral disc under compression, to understand the damage initiation and propagation. It is found that, for this case (with 1000N/s of compression rate and neglected ground matrix damage), the damage initiated in the outer and middle posterior regions of AF at about 700N of axial compression. The region-dependent yield stretch ratio predicted by this model is consistent with experimental findings. A sensitive study on the damage parameters is also presented. This study provides an additional insight into AF damage in the isolated disc under mechanical compression.

MR Elastography–derived Stiffness: A Biomarker for Intervertebral Disc Degeneration

Purpose To determine the repeatability of magnetic resonance (MR) elastography-derived shear stiffness measurements of the intervertebral disc (IVD) taken throughout the day and their relationship with IVD degeneration and subject age. Materials and Methods In a cross-sectional study, in vivo lumbar MR elastography was performed once in the morning and once in the afternoon in 47 subjects without current low back pain (IVDs = 230; age range, 20-71 years) after obtaining written consent under approval of the institutional review board. The Pfirrmann degeneration grade and MR elastography-derived shear stiffness of the nucleus pulposus and annulus fibrosus regions of all lumbar IVDs were assessed by means of principal frequency analysis. One-way analysis of variance, paired t tests, concordance and Bland-Altman tests, and Pearson correlations were used to evaluate degeneration, diurnal changes, repeatability, and age effects, respectively. Results There were no significant differences between morning and afternoon shear stiffness across all levels and there was very good technical repeatability between the morning and afternoon imaging results for both nucleus pulposus (R = 0.92) and annulus fibrosus (R = 0.83) regions. There was a significant increase in both nucleus pulposus and annulus fibrosus MR elastography-derived shear stiffness with increasing Pfirrmann degeneration grade (nucleus pulposus grade 1, 12.5 kPa ± 1.3; grade 5, 16.5 kPa ± 2.1; annulus fibrosus grade 1, 90.4 kPa ± 9.3; grade 5, 120.1 kPa ± 15.4), and there were weak correlations between shear stiffness and age across all levels (R ≤ 0.32). Conclusion Our results demonstrate that MR elastography-derived shear stiffness measurements are highly repeatable, weakly correlate with age, and increase with advancing IVD degeneration. These results suggest that MR elastography-derived shear stiffness may provide an objective biomarker of the IVD degeneration process. © RSNA, 2017 Online supplemental material is available for this article.

Hybrid Rigid-Deformable Model for Prediction of Neighboring Intervertebral Disk Loads During Flexion Movement After Lumbar Interbody Fusion at L3-4 Level.

Knowledge of spinal loads in neighboring disks after interbody fusion plays an important role in the clinical decision of this treatment as well as in the elucidation of its effect. However, controversial findings are still noted in the literature. Moreover, there are no existing models for efficient prediction of intervertebral disk stresses within annulus fibrosus (AF) and nucleus pulposus (NP) regions. In this present study, a new hybrid rigid-deformable modeling workflow was established to quantify the mechanical stress behaviors within AF and NP regions of the L1-2, L2-3, and L4-5 disks after interbody fusion at L3-4 level. The changes in spinal loads were compared with results of the intact model without interbody fusion. The fusion outcomes revealed maximal stress changes (10%) in AF region of L1-2 disk and in NP region of L2-3 disk. The minimal stress change (1%) is noted at the NP region of the L1-2 disk. The validation of simulation outcomes of fused and intact lumbar spine models against those of other computational models and in vivo measurements showed good agreements. Thus, this present study may be used as a novel design guideline for a specific implant and surgical scenario of the lumbar spine disorders.

Enhanced Musculoskeletal Modeling for Prediction of Intervertebral Disc Stress Within Annulus Fibrosus and Nucleus Pulposus Regions During Flexion Movement

Classical rigid musculoskeletal lumbar spine models cannot provide spinal load within intervertebral discs (IVDs) due to modeling assumptions. The present study develops an approach for predicting IVD stress in annulus fibrosus (AF) and nucleus pulposus (NP) regions. A musculoskeletal model coupled with elastic elements placed between vertebrae is developed. These elements are distributed within AF and NP regions of each IVD. Each elastic element representing a standard nonlinear solid model allows vertebral strain-like displacement and IVD stress during flexion motion to be computed. Maximal stresses at the L4-5 level are 1.25 and 1.4 MPa for AF and NP regions, respectively, during 40° flexion motion. Maximal stresses at the L2-3, L1-2, and L3-4 levels are 1.93 and 2.2 MPa, 1.12 and 1.2 MPa, and 2.68 MPa for AF and NP regions, respectively. Vertebral displacements, muscle forces, and predicted stresses are in agreement with data reported in the literature. This study opens new perspectives for further efficient musculoskeletal spine models for predicting spinal loads, leading to improved and optimized treatment, recovery, and follow-up recommendations for spinal disorders.

Link N as a Therapeutic Agent to Treat Pain Associated with Intervertebral Disc Degeneration

Introduction The intervertebral discs (IVDs) are the largest avascular tissues in the body characterized by an abundant extracellular matrix and low cell density, with innervation limited at the most peripheral annular layers. During IVD degeneration, the neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), can stimulate nerve growth and hyperinnervation of the inner annulus fibrosus (AF) and nucleus pulposus (NP), which further could lead to back pain. Back pain can potentially be suppressed by BMP4 supplementation to decrease the density of innervation. Link N is a naturally occurring peptide that can stimulate BMP4 in human IVDs. The aim of the present study was to determine the effect of Link N on NGF, BDNF, and substance P (SP) in human AF cells stimulated with cytokines or punctured bovine discs and determine the possible mechanisms related to its effect. Material and Methods Human lumbar spines were obtained through Héma-Québec harvesting organ donation within 24 hour after death. Annulus fibrosus (AF) cells were isolated from AF region of human IVDs with Thompson grade 2 of degeneration, cultured in monolayers and stimulated with TNFα (100 ng/mL) and IL1β (10 ng/mL) in the presence or absence of Link N (1 µg/mL) for 48 hours. Total RNA was isolated and gene expression of neurotrophins, substance P, and their receptors was measured using RT PCR. AF cells isolated from human IVDs with Thompson grade 4 were cultured for 48 hours with or without Link N (1 µg/mL) and the NGF release in the media was measured by Western blotting. Coccygeal IVDs from the tails of adult bovine steers (20–25 months) were isolated with the cartilaginous endplates. After 24 hours preconditioning in complete DMEM, the IVDs were treated (control, capsaicin [1.5 µg/mL), punctured by 16 G needle, punctured by 16 G needle and incubated with Link N [10 µg/mL]) and the disc culture media was collected at different time points for analysis. SP in the media was concentrated by solid phase extraction and was assayed by Elisa. Results Proinflammatory cytokines IL1β and TNFα have been shown to trigger the expression of NGF and SP, as well as promoting the degradation of the extracellular matrix in human IVD cells. Our results demonstrate that gene expression of NGF, BDNF, TrkA, and TrkB significantly decreased in human disc cells stimulated with either IL-1β or TNFα supplemented with Link N when compared with the control. Additionally, Link N was also able to inhibit NGF release in the media when AF grade 4 was stimulated with IL-1β. Link N supplementation also led to a significant decrease in SP after 24 hour in injured discs, when compared with controls. Conclusion Link N can suppress the stimulation of NGF, BDNF, and their receptors TrkA and TrkB, respectively, as well as the neurotransmitter SP in degenerate discs. This suggests that Link N has the potential to inhibit pain induced by neuronal innervation caused by disc degeneration. Thus, administration of Link N has the potential to not only repair the discs in early stages of the disease but also suppress pain.

Dose-dependent response of tissue-engineered intervertebral discs to dynamic unconfined compressive loading.

Because of the limitations of current surgical methods in the treatment of degenerative disc disease, tissue-engineered intervertebral discs (TE-IVDs) have become an important target. This study investigated the biochemical and mechanical responses of composite TE-IVDs to dynamic unconfined compression. TE-IVDs were manufactured by floating an injection molded alginate nucleus pulposus (NP) in a type I collagen annulus fibrosus (AF) that was allowed to contract for 2 weeks before loading. The discs were mechanically stimulated at a range of strain amplitude (1-10%) for 2 weeks with a duty cycle of 1 h on-1 h off-1 h on before being evaluated for their biochemical and mechanical properties. Mechanical loading increased all properties in a dose-dependent manner. Glycosaminoglycans (GAGs) increased between 2.8 and 2.2 fold in the AF and NP regions, respectively, whereas the hydroxyproline content increased between 1.2 and 1.8 fold. The discs also experienced a 2-fold increase in the equilibrium modulus and a 4.3-fold increase in the instantaneous modulus. Full effects for all properties were seen by 5% strain amplitude. These data suggest that dynamic loading increases the functionality of our TE-IVDs with region-dependent responses using a method that may be scaled up to larger disc models to expedite maturation for implantation.

Temporal changes of mechanical signals and extracellular composition in human intervertebral disc during degenerative progression

In this study, a three-dimensional finite element model was used to investigate the changes in tissue composition and mechanical signals within human lumbar intervertebral disc during the degenerative progression. This model was developed based on the cell-activity coupled mechano-electrochemical mixture theory. The disc degeneration was simulated by lowering nutrition levels at disc boundaries, and the temporal and spatial distributions of the fixed charge density, water content, fluid pressure, Von Mises stress, and disc deformation were analyzed. Results showed that fixed charge density, fluid pressure, and water content decreased significantly in the nucleus pulposus (NP) and the inner to middle annulus fibrosus (AF) regions of the degenerative disc. It was found that, with degenerative progression, the Von Mises stress (relative to that at healthy state) increased within the disc, with a larger increase in the outer AF region. Both the disc volume and height decreased with the degenerative progression. The predicted results of fluid pressure change in the NP were consistent with experimental findings in the literature. The knowledge of the variations of temporal and spatial distributions of composition and mechanical signals within the human IVDs provide a better understanding of the progression of disc degeneration.