Human Lumbar Discs Research Articles

Contribution of facet joints, axial compression, and composition to human lumbar disc torsion mechanics.

Stresses applied to the spinal column are distributed between the intervertebral disc and facet joints. Structural and compositional changes alter stress distributions within the disc and between the disc and facet joints. These changes influence the mechanical properties of the disc joint, including its stiffness, range of motion, and energy absorption under quasi-static and dynamic loads. There have been few studies evaluating the role of facet joints in torsion. Furthermore, the relationship between biochemical composition and torsion mechanics is not well understood. Therefore, the first objective of this study was to investigate the role of facet joints in torsion mechanics of healthy and degenerated human lumbar discs under a wide range of compressive preloads. To achieve this, each disc was tested under four different compressive preloads (300-1200 N) with and without facet joints. The second objective was to develop a quantitative structure-function relationship between tissue composition and torsion mechanics. Facet joints have a significant contribution to disc torsional stiffness (∼60%) and viscoelasticity, regardless of the magnitude of axial compression. The findings from this study demonstrate that annulus fibrosus GAG content plays an important role in disc torsion mechanics. A decrease in GAG content with degeneration reduced torsion mechanics by more than an order of magnitude, while collagen content did not significantly influence disc torsion mechanics. The biochemical-mechanical and compression-torsion relationships reported in this study allow for better comparison between studies that use discs of varying levels of degeneration or testing protocols and provide important design criteria for biological repair strategies. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Temporal and spatial variations of pressure within intervertebral disc nuclei.

Experimental and computational studies often presume that nuclei pulposi of non-degenerated human lumbar discs function as fluid-filled cavities with single hydrostatic pressures throughout that vary neither with time nor location and orientation. Recent simultaneous measurements of the pressure at multiple locations within disc nuclei have however shown time-dependent and nonhomogeneous pressure distributions. This combined in vitro and in silico study aims to re-examine the temporal and spatial variations of the pressure within disc nuclei with special focus on the effect of tissue hydration. After 20h of free swelling, effects of two preload magnitudes (0.06 and 0.28MPa) on nucleus pressure were investigated under 8h of constant preloads followed by 10 cycles of high-low loads each lasting 15min using 6 disc-bone bovine specimens. Changes in pressure at 3 different nucleus locations were recorded as surrogate measures of fluid flow within the discs. To identify the likely mechanisms observed in vitro, a finite element model of a human disc (L4-L5) was employed while simulating foregoing plus additional loading protocols. In vitro and computed results show a clear and substantial pressure gradient within the nucleus, especially early after the load application under higher loads and in more hydrated discs. The pressure reaches its maximum in the nucleus center reducing axially toward endplates and radially toward the nucleus-annulus interface. These cause pressure gradients that substantially diminish with time and at lower hydration levels. With time and as the pore pressure drops, the contribution of the nucleus bulk increases till it reaches equilibrium. The relative share of the annulus bulk in supporting the applied loads markedly increases not only with time but at higher loads and lower hydrations. The hydration state of the disc is hence crucial in the disc pressure distribution and internal response under various static-dynamic loads in vitro and in the replication of in vivo conditions.

Role of miR-589-3p in human lumbar disc degeneration and its potential mechanism.

The present study aimed to investigate the role of miR-589-3p in lumbar disc degeneration (LDD) and to explore the underlying mechanisms. Nucleus pulposus (NP) cells were stimulated with lipopolysaccharide (LPS) to simulate an in vitro model of intervertebral disc degeneration. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression level of microRNA (miR)-589-3p in the NP cells, and the results demonstrated that the increased expression of miR-589-3p in LPS stimulated NP cells compared with the control. To further investigate the role of miR-589-3p in LDD, a human NP cell line with high/low miR-589-3p expression was generated using miR-589-3p mimics/inhibitors. In addition, a human NP cell inflammation model was conducted by LPS (10 µM) treatment. Western blot analysis and RT-qPCR were performed for detection of associated genes and proteins. Protein levels of pro-inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 were evaluated by ELISA. Flow cytometry was used for cell apoptosis determination. Furthermore, Targetscan was used to predict potential targets of miR-589-3p, and a dual luciferase reporter assay was used to verify the prediction. The findings verified that miR-589-3p was significantly upregulated in LDD. In vitro, miR-589-3p mimics/inhibitors significantly increased/reduced the production of TNF-α, IL-1β and IL-6 in LPS stimulated NP cells. Furthermore, miR-589-3p mimics/inhibitors significantly promoted/inhibited LPS stimulated NP cell apoptosis. MiR-589-3p mimics/inhibitors significantly repressed/enhanced type II collagen and aggrecan expression in LPS stimulated NP cells. In addition, it was demonstrated that mothers against decapentaplegic homolog (Smad) 4 was a direct target gene of miR-589-3p, and was negatively regulated by miR-589-3p in NP cells. In conclusion, miR-589-3p may function as a promoter in LDD development via the regulation of Smad4.

Human and bovine spinal disc mechanics subsequent to trypsin injection

ObjectiveTo investigate the biomechanical effects of injections of a protease on the characteristics of bovine coccygeal and human lumbar disc motion segments. MethodsMechanics of treated tissues were measured immediately after injection and 3 h after injection. Motion segments underwent axial rotation and flexion-extension loading. ResultsStiffness and neutral zone parameters experienced significant changes over time, with bovine tissues more strongly affected than human cadaver tissues. This was true in both axial rotation and flexion-extension. The treatment type significantly affected the neutral zone measurements in axial rotation. Hysteresis parameters were impacted by control injections. ConclusionThe extrapolation of bovine coccygeal motion testing results to human lumbar disc mechanics is not yet practical. The injected treatment may have a smaller impact on disc mechanics than time in testing. Viscoelasticity of human lumbar discs may be impacted by any damage to the annulus fibrosis induced by needlestick. The Translational Potential of this ArticlePreclinical testing of novel spinal devices is essential to the design validation and regulatory processes, but current testing techniques rely on cadaveric testing of primarily older spines with essentially random amounts of disc degeneration. The present work investigates the viability of using trypsin injections to create a more uniform preclinical model of disc degeneration from a mechanics perspective, for the purpose of testing spinal devices. Such a model would facilitate translation of new spinal technologies to clinical practice.

Oxidative damage induces apoptosis and promotes calcification in disc cartilage endplate cell through ROS/MAPK/NF-κB pathway: Implications for disc degeneration

Cartilage endplate (CEP) cell calcification and apoptosis play a vital role in the intervertebral disc degeneration (IVDD). Oxidative stress is a key factor in inducing programmed cell death and cartilage calcification. However, the cell death and calcification of cartilage endplate cells under oxidative stress have never been described. The present study investigated the apoptosis and calcification in the cartilage endplate cell under oxidative stress induced by H2O2 to understand the underlying mechanism of IVDD. The cartilage endplate cells isolated from human lumbar discs were subjected to different concentrations of H2O2 for various time periods. The cell viability was determined by CCK-8 assay, whereas Western blot, immunofluorescence, and Alcian blue, Alizarin red, and Von Kossa staining evaluated the apoptosis and calcification. The level of mitochondria-specific reactive oxygen species (ROS) was quantified with an oxygen radical-sensitive probe—MitoSOX. The potential signaling pathways were investigated by Western blot after the addition of N-acetyl-l-cysteine (NAC). We found that the oxidative stress induced by H2O2 increased the apoptosis and subsequently the calcification in the cartilage endplate cells through the ROS/p38/ERK/p65 pathway. The apoptosis and the calcification of the cartilage endplate cells induced by H2O2 can be abolished by NAC. These results suggested that regulating the apoptosis and the calcification in the cartilage endplate cells under oxidative stress should be advantageous for the survival of cells and might delay the process of disc degeneration.

ISSLS PRIZE IN CLINICAL SCIENCE 2017: Is infection the possible initiator of disc disease? An insight from proteomic analysis.

Proteomic and 16S rDNA analysis of disc tissues obtained in vivo. To address the controversy of infection as an aetiology for disc disorders through protein profiling. There is raging controversy over the presence of bacteria in human lumbar discs in vivo, and if they represent contamination or infection. Proteomics can provide valuable insight by identifying proteins signifying bacterial presence and, also host defence response proteins (HDRPs), which will confirm infection. 22 discs (15-disc herniations (DH), 5-degenerate (DD), 2-normal in MRI (NM) were harvested intraoperatively and immediately snap frozen. Samples were pooled into three groups and proteins extracted were analysed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Post identification, data analysis was performed using Uniprotdb, Pantherdb, Proteome discoverer and STRING network. Authentication for bacterial presence was performed by PCR amplification of 16S rDNA. LC-MS/MS analysis using Orbitrap showed 1103 proteins in DH group, compared to 394 in NM and 564 in DD. 73 bacterial specific proteins were identified (56 specific for Propionibacterium acnes; 17 for Staphylococcus epidermidis). In addition, 67 infection-specific HDRPs, unique or upregulated, such as Defensin, Lysozyme, Dermcidin, Cathepsin-G, Prolactin-Induced Protein, and Phospholipase-A2, were identified confirming presence of infection. Species-specific primers for P. acnes exhibited amplicons at 946bp (16S rDNA) and 515bp (Lipase) confirming presence of P. acnes in both NM discs, 11 of 15 DH discs, and all five DD discs. Bioinformatic search for protein-protein interactions (STRING) documented 169 proteins with close interactions (protein clustering co-efficient 0.7) between host response and degenerative proteins implying that infection may initiate degradation through Ubiquitin C. Our study demonstrates bacterial specific proteins and host defence proteins to infection which strengthen the hypothesis of infection as a possible initiator of disc disease. These results can lead to a paradigm shift in our understanding and management of disc disorders.

Landscape of RNAs in human lumbar disc degeneration.

Accumulating evidence indicates noncoding RNAs (ncRNAs) fine-tune gene expression with mysterious machinery. We conducted a combination of mRNA, miRNA, circRNA, LncRNA microarray analyses on 10 adults' lumbar discs. Moreover, we performed additional global exploration on RNA interacting machinery in terms of in silico computational pipeline. Here we show the landscape of RNAs in human lumbar discs. In general, the RNA-abundant landscape comprises 14,635 mRNAs (37.93%), 2,059 miRNAs (5.34%), 18,995 LncRNAs (49.23%) and 2,894 (7.5%) circRNAs. Chromosome 1 contributes for RNA transcription at most (10%). Bi-directional transcription contributes evenly for RNA biogenesis, in terms of 5′ to 3′ and 3′ to 5′. Despite the majority of circRNAs are exonic, antisense (1.49%), intergenic (0.035%), intragenic (1.69%), and intronic (6.29%) circRNAs should not be ignored. A single miRNA could interact with a multitude of circRNAs. Notably, CDR1as or ciRS-7 harbors 66 consecutive binding sites for miR-7-5p (previous miR-7), evidencing our pipeline. The majority of binding sites are perfect-matched (78.95%). Collectively, global landscape of RNAs sheds novel insights on RNA interacting mechanisms in human intervertebral disc degeneration.

Kinetics of charged antibiotic penetration into human intervertebral discs: A numerical study

Little quantitative information exists on the kinetics of charged antibiotic penetration into human intervertebral discs (IVD). This information is crucial for determining the dosage to use, timing of administration, and duration of treatment for infected IVDs. The objective of this study was to quantitatively analyze the transport of various charged antibiotics into human lumbar IVDs. Penetration of charged and uncharged antibiotics into a human lumbar disc was analyzed using a 3D finite element model. The valence (z) of the electrical charge of antibiotics varied from z=+2 (positively charged) to z=−2 (negatively charged). An uncharged antibiotic (z=0) was used as a control. Cases with intravenous (IV) administrations of different charged antibiotics were simulated. Our results showed that the electrical charge had great effects on kinetics of an antibiotic penetration into the IVD; with higher concentrations and uptakes for positively charged antibiotics than those for negatively charged ones. This study provides quantitative information on selecting antibiotics for treating intervertebral disc infections.

Effect of Annular Defects on Intradiscal Pressures in the Lumbar Spine: An in Vitro Biomechanical Study of Diskectomy and Annular Repair.

Background Integrity of intervertebral disks may influence, and be influenced by, the maintenance of hydrostatic pressures inside the nucleus pulposus. Disk degeneration causes decreased pressures, leading to overload and injury of the annulus fibrosus, increasing the risk of disk herniation. Diskectomies to treat disk herniation can cause further loss of hydrostatic pressures resulting in worsening degeneration. This study investigated the impact of opening the annulus on intradiscal pressure and whether implantation of an annular closure device (ACD) can restore physiologic pressures. Methods The pressure responses under unconstrained moments in concert with axial compressive loads of nine human cadaver lumbar disks were biomechanically tested at baseline, immediately following posterior annulotomy, and immediately following implantation of the ACD. Results The analysis of variance indicated a significant difference in the pressure response (p = 0.0001) among the three rounds of testing. Specifically, the post hoc Bonferroni test revealed that the pressure response after diskectomy was significantly different when compared with baseline (p < 0.001) and after ACD implantation (p = 0.001). However, baseline and ACD pressure responses were insignificantly different (p = 1.000). Conclusion Our findings suggest that restoration of annular integrity during diskectomy with implantation of the tested ACD may restore pressures closer to preoperative levels. Whether or not restoring pressures to preoperative levels has any clinical benefit or effect on the rate of degeneration is an area for further clinical research.

Assessing the correlation between the degree of disc degeneration on the Pfirrmann scale and the metabolites identified in HR-MAS NMR spectroscopy

ObjectiveThe objective of this study is to assess the correlation between the degree of degeneration of lumbar discs according to the Pfirrmann classification system and the concentrations of metabolites determined by means of 1H high-resolution magic angle spinning nuclear magnetic resonance (1H HR MAS NMR) spectroscopy. Materials and methodsTwenty-six human intervertebral lumbar discs that were operated on due to degenerative disease were analyzed. Routine preoperative 1.5T, T2-weighed magnetic resonance (MR) images were used to classify the cases according to the Pfirrmann classification system. In all the cases, during microdiscectomy, the fragments of the annulus fibrosus and nucleus pulposus were harvested and their metabolic profile was examined by means of 1H HR MAS. The grades of disc degeneration on the Pfirrmann scale were correlated with the metabolite concentrations. ResultsSpectral analyses of the intervertebral discs with Pfirrmann grades IV and V demonstrated significantly higher concentrations of creatine, glycine, hydroxyproline, alanine, leucine, valine, acetate, isoleucine, α,β-glucose, and myo-inositol, and a lower intensity of the N-acetyl peak of chondroitin sulfate, compared to the spectra with Pfirrmann grade III. ConclusionOur results demonstrate correlations between metabolite concentrations and the degree of lumbar disc degeneration assessed using the Pfirrmann grading system and provide another step toward the potential use of in vivo MR spectroscopy for investigation of biomarkers in lumbar disc degeneration.

Biomechanical analysis of the camelid cervical intervertebral disc

SummaryChronic low back pain (LBP) is a prevalent global problem, which is often correlated with degenerative disc disease. The development and use of good, relevant animal models of the spine may improve treatment options for this condition. While no animal model is capable of reproducing the exact biology, anatomy, and biomechanics of the human spine, the quality of a particular animal model increases with the number of shared characteristics that are relevant to the human condition. The purpose of this study was to investigate the camelid (specifically, alpaca and llama) cervical spine as a model of the human lumbar spine. Cervical spines were obtained from four alpacas and four llamas and individual segments were used for segmental flexibility/biomechanics and/or morphology/anatomy studies. Qualitative and quantitative data were compared for the alpaca and llama cervical spines, and human lumbar specimens in addition to other published large animal data. Results indicate that a camelid cervical intervertebral disc (IVD) closely approximates the human lumbar disc with regard to size, spinal posture, and biomechanical flexibility. Specifically, compared with the human lumbar disc, the alpaca and llama cervical disc size are approximately 62%, 83%, and 75% with regard to area, depth, and width, respectively, and the disc flexibility is approximately 133%, 173%, and 254%, with regard to range of motion (ROM) in axial-rotation, flexion-extension, and lateral-bending, respectively. These results, combined with the clinical report of disc degeneration in the llama lower cervical spine, suggest that the camelid cervical spine is potentially well suited for use as an animal model in biomechanical studies of the human lumbar spine.

Nonlinear dynamics of the human lumbar intervertebral disc

Systems with a quasi-static response similar to the axial response of the intervertebral disc (i.e. progressive stiffening) often present complex dynamics, characterized by peculiar nonlinearities in the frequency response. However, such characteristics have not been reported for the dynamic response of the disc. The accurate understanding of disc dynamics is essential to investigate the unclear correlation between whole body vibration and low back pain. The present study investigated the dynamic response of the disc, including its potential nonlinear response, over a range of loading conditions. Human lumbar discs were tested by applying a static preload to the top and a sinusoidal displacement at the bottom of the disc. The frequency of the stimuli was set to increase linearly from a low frequency to a high frequency limit and back down. In general, the response showed nonlinear and asymmetric characteristics. For each test, the disc had different response in the frequency-increasing compared to the frequency-decreasing sweep. In particular, the system presented abrupt changes of the oscillation amplitude at specific frequencies, which differed between the two sweeps. This behaviour indicates that the system oscillation has a different equilibrium condition depending on the path followed by the stimuli. Preload and amplitude of the oscillation directly influenced the disc response by changing the nonlinear dynamics and frequency of the jump-phenomenon. These results show that the characterization of the dynamic response of physiological systems should be readdressed to determine potential nonlinearities. Their direct effect on the system function should be further investigated.

In vivo morphological features of human lumbar discs.

Recent biomechanics studies have revealed distinct kinematic behavior of different lumbar segments. The mechanisms behind these segment-specific biomechanical features are unknown. This study investigated the in vivo geometric characteristics of human lumbar intervertebral discs.Magnetic resonance images of the lumbar spine of 41 young Chinese individuals were acquired. Disc geometry in the sagittal plane was measured for each subject, including the dimensions of the discs, nucleus pulposus (NP), and annulus fibrosus (AF). Segmental lordosis was also measured using the Cobb method.In general, the disc length increased from upper to lower lumbar levels, except that the L4/5 and L5/S1 discs had similar lengths. The L4/5 NP had a height of 8.6 ± 1.3 mm, which was significantly higher than all other levels (P < 0.05). The L5/S1 NP had a length of 21.6 ± 3.1 mm, which was significantly longer than all other levels (P < 0.05). At L4/5, the NP occupied 64.0% of the disc length, which was significantly less than the NP of the L5/S1 segment (72.4%) (P < 0.05). The anterior AF occupied 20.5% of the L4/5 disc length, which was significantly greater than that of the posterior AF (15.6%) (P < 0.05). At the L5/S1 segment, the anterior and posterior AFs were similar in length (14.1% and 13.6% of the disc, respectively). The height to length (H/L) ratio of the L4/5 NP was 0.45 ± 0.06, which was significantly greater than all other segments (P < 0.05). There was no correlation between the NP H/L ratio and lordosis.Although the lengths of the lower lumbar discs were similar, the geometry of the AF and NP showed segment-dependent properties. These data may provide insight into the understanding of segment-specific biomechanics in the lower lumbar spine. The data could also provide baseline knowledge for the development of segment-specific surgical treatments of lumbar diseases.

Upregulation of BDNF and NGF in cervical intervertebral discs exposed to painful whole-body vibration.

In vivo study defining expression of the neurotrophins, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), in cervical intervertebral discs after painful whole-body vibration (WBV). The goal of this study is to determine if BDNF and NGF are expressed in cervical discs after painful WBV in a rat model. WBV is a possible source of neck pain and has been implicated as increasing the risk for disc disorders. Typically, aneural regions of painful human lumbar discs exhibit hyperinnervation, suggesting nerve ingrowth as potentially contributing to disc degeneration and pain. BDNF and NGF are upregulated in painfully degenerate lumbar discs and hypothesized to contribute to this pathology. Male Holtzman rats underwent 7 days of repeated WBV (15 Hz, 30 min/d) or sham exposures, followed by 7 days of rest. Cervical discs were collected for analysis of BDNF and NGF expression through RT-qPCR and Western blot analysis. Immunohistochemistry also evaluated their regional expression in the disc. Vibration significantly increases BDNF messenger ribonucleic acid (mRNA) levels (P=0.036), as well as total-NGF mRNA (P=0.035). Protein expression of both BDNF (P=0.006) and the 75-kDa NGF (P=0.045) increase by nearly 4- and 10-fold, respectively. Both BDNF mRNA (R=0.396; P=0.012) and protein (R=0.280; P=0.035) levels are significantly correlated with the degree of behavioral sensitivity (i.e., pain) at day 14. Total-NGF mRNA is also significantly correlated with the extent of behavioral sensitivity (R=0.276; P=0.044). Both neurotrophins are most increased in the inner annulus fibrosus and nucleus pulposus. The increases in BDNF and NGF in the cervical discs after painful vibration are observed in typically aneural regions of the disc, consistent with reports of its hyperinnervation. Yet, the induction of nerve ingrowth into the disc was not explicitly investigated. Neurotrophin expression also correlates with behavioral sensitivity, suggesting a role for both neurotrophins in the development of disc pain. N/A.

Intervertebral disc regeneration: do nutrients lead the way?

Strategies for the biological repair of intervertebral discs derive from the premise that disc degeneration results from impaired cellular activity and, therefore, that these structures can be induced to regenerate by implanting active cells or providing factors that restore normal cellular activity. In vitro and animal studies using this approach have had some success, but whether this success can be reproduced in degenerate human lumbar discs is unknown. Successful repair requires that the disc cells remain viable and active; they therefore need an adequate supply of nutrients. However, as the disc degenerates, the nutrient supply decreases, thereby limiting cell activity and viability. Current biologic approaches might place additional demands on an already precarious nutrient supply. Here, we discuss whether the loss of nutrients associated with disc degeneration limits the effectiveness of biologic approaches, and indicate that this neglected problem requires investigation if clinical application of such therapies is to succeed.

MR diffusion is sensitive to mechanical loading in human intervertebral disks ex vivo.

To use T2 and diffusion MR to determine the change in the mechanical function of human disks with increased degenerative state. Spatial changes in T2 and diffusion were quantified in five cadaveric human lumbar disks under compressive loads. Regression models were used to investigate the relationship between the change in MR parameters and the disk's dynamic and viscoelastic properties. Compressive loading caused a significant reduction in the disk's mean diffusivity ([11.3 versus 9.7].10(-4) .mm(2) /s, P < 0.001) but little change in T2 (P < 0.05). Diffusivity and T2 were correlated with the disk's dynamic (P < 0.01 and P < 0.05) and long-term viscoelastic (P < 0.05 and P < 0.05) stiffness. Diffusivity but not T2, was correlated with its viscoelastic dampening (r(2) = 0.45, P < 0.01) and instantaneous stiffness (r(2) = 0.44, P < 0.05). Nucleus diffusivity was significantly higher than the annulus's (-21% to -4%, P < 0.01). MR-estimated hydration was correlated with the instantaneous viscoelastic stiffness of the nucleus (r(2) = 0.35, P < 0.05) and the dynamic (r(2) = 0.44, P < 0.05) and long-term viscoelastic (r(2) = 0.42, P < 0.05) stiffness in the annulus. T2 correlated with diffusivity at low load (r(2) = 0.66, P < 0.05), but not at high load. The strong correlations between diffusivity and the rheological assessments of disk mechanics suggest that MR might permit quantitative assessment of disk functional status and structural integrity.

New Insight into Human Disc Degeneration by Gene Expression Profiling

Introduction Intervertebral disc (IVD) degeneration is characterized by the breakdown of extracellular matrix molecules and is often associated with inflammatory processes. Hence, anabolic, anti-catabolic, or anti-inflammatory treatments have been considered to retard or reverse early degenerative changes in the IVD. However, there is still a lack of fundamental knowledge about the molecular transformations in the degenerative compared with the native healthy discs. More detailed understanding of the molecular mechanisms will be essential to develop specific therapeutic strategies. In the present study, microarray and quantitative gene expression analysis were used to compare expression profiles of cells from healthy and degenerative human IVDs. The aim is to identify significantly dysregulated molecules or pathways that can potentially be targeted for regenerative therapy. Materials and Methods Annulus fibrosus (AF) and nucleus pulposus (NP) tissues were obtained from human lumbar discs through organ donation program and in accordance with the local and institutional ethical guidelines. Harvested tissue was assigned to either the “healthy” (Thompson disc degeneration grade I-II) or the “degenerative” group (Thompson grade III-IV). Cells were isolated from tissues using sequential pronase and collagenase digestion. Total RNA was extracted from isolated cells using a TRI-Spin method. Samples were processed and subjected to Affymetrix Whole Human Genome DNA microarray profiling. After correspondence analysis for data correction, expression differences between healthy and degenerative AF and NP cells, respectively, were analyzed. Genes with most significant expression divergences were further assessed using quantitative real time RT-PCR. Results For both NP and AF, n = 8 healthy and n = 16 degenerative RNA samples were profiled by microarray. Biostatistical analysis revealed that 237 genes were differentially regulated between degenerative and healthy human AF cells, while 178 genes were differentially regulated between degenerative and healthy NP cells. Specifically, 119 (AF) and 66 (NP) genes were found up-regulated, whereas 118 (AF) and 112 (NP) genes were down-regulated in the degenerative group. Quantitative gene expression analysis was performed for the most significantly differentially regulated genes in n = 8 healthy and n = 10 degenerative RNA samples. While some genes were specifically modulated in the AF or NP cells, several genes showed significant differential regulation ( p &lt; 0.05) by degeneration status. Genes up-regulated in degenerative compared with healthy IVD cells included activated leukocyte cell adhesion molecule (ALCAM), cyclin D1 (CCND1), insulin-like growth factor binding protein 3 (IGFBP3), interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), magnesium transporter 1 (MAGT1), and tissue factor pathway inhibitor (TFPI). Significantly down-regulated genes in degenerative compared with healthy IVD cells, include dickkopf 1 homolog (DKK1), forkhead box F2 (FOXF2), lectin galactoside-binding-like (LGALSL), lipoprotein lipase (LPL), and mannosidase (MAN2B2). Moreover, modulation of distinct molecular pathways could be recognized. Conclusion The present data demonstrates upregulation of signaling factors and antagonists, growth factors and their inhibitors, and chemotactic factors in degenerative cells. Moreover, factors involved in matrix turnover were down-regulated. Improved insight in the differential regulation of distinct molecules and pathways may ultimately allow specific treatment on a molecular basis. Further validation at the protein expression level will be required to evaluate their potential as therapeutic target or as biomarkers for predicting the state of degenerative process in the discs. Disclosure of Interest S. Grad: Conflict with AO Foundation Collaborative Research Program Annulus Fibrosus Repair R. Gawri: None declared L. Haglund: None declared J. Ouellet: Conflict with DePuySynthes, AO Foundation, AONA F. Mwale: None declared L. Creemers: Conflict with Dutch Arthritis Association J. Rutges: None declared W. Gallagher: None declared P. O'Gaora: None declared Pandit: None declared M. Alini: Conflict with AO Foundation Collaborative Research Program Annulus Fibrosus Repair

Quantitative T2* (T2 star) relaxation times predict site specific proteoglycan content and residual mechanics of the intervertebral disc throughout degeneration.

Degeneration alters the biochemical composition of the disc, affecting the mechanical integrity leading to spinal instability. Quantitative T2* MRI probes water mobility within the macromolecular network, a potentially more sensitive assessment of disc health. We determined the relationship between T2* relaxation time and proteoglycan content, collagen content, and compressive mechanics throughout the degenerative spectrum. Eighteen human cadaveric lumbar (L4-L5) discs were imaged using T2* MRI. The T2* relaxation time at five locations (nucleous pulposus or NP, anterior annulus fibrosis or AF, posterior AF, inner AF, and outer AF) was correlated with sulfated-glycosaminoglycan (s-GAG) content, hydroxyproline content, and residual stress and strain at each location. T2* relaxation times were significantly correlated with s-GAG contents in all test locations and were particularly strong in the NP (r = 0.944; p < 0.001) and inner AF (r = 0.782; p < 0.001). T2* relaxation times were also significantly correlated with both residual stresses and excised strains in the NP (r = 0.857; p < 0.001: r = 0.816; p < 0.001), inner AF (r = 0.535; p = 0.022: r = 0.516; p = 0.028), and outer AF (r = 0.668; p = 0.002: r = 0.458; p = 0.041). These strong correlations highlight T2* MRI's ability to predict the biochemical and mechanical health of the disc. T2* MRI assessment of disc health is a clinically viable tool showing promise as a biomarker for distinguishing degenerative changes.

Effect of Link N on the Expression of Neurotrophins and Substance P Release by Human Intervertebral Disc Cells Stimulated with Proinflammatory Cytokines

IntroductionAlthough there are different causes of low back pain, intervertebral disc (IVD) degeneration is one of them. In healthy discs, nociceptive nerve fibers and mechanoreceptors penetrate up...

Elastic, permeability and swelling properties of human intervertebral disc tissues: A benchmark for tissue engineering

The aim of functional tissue engineering is to repair and replace tissues that have a biomechanical function, i.e., connective orthopaedic tissues. To do this, it is necessary to have accurate benchmarks for the elastic, permeability, and swelling (i.e., biphasic-swelling) properties of native tissues. However, in the case of the intervertebral disc, the biphasic-swelling properties of individual tissues reported in the literature exhibit great variation and even span several orders of magnitude. This variation is probably caused by differences in the testing protocols and the constitutive models used to analyze the data. Therefore, the objective of this study was to measure the human lumbar disc annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplates (CEP) biphasic-swelling properties using a consistent experimental protocol and analyses. The testing protocol was composed of a swelling period followed by multiple confined compression ramps. To analyze the confined compression data, the tissues were modeled using a biphasic-swelling model, which augments the standard biphasic model through the addition of a deformation-dependent osmotic pressure term. This model allows considering the swelling deformations and the contribution of osmotic pressure in the analysis of the experimental data. The swelling stretch was not different between the disc regions (AF: 1.28±0.16; NP: 1.73±0.74; CEP: 1.29±0.26), with a total average of 1.42. The aggregate modulus (Ha) of the extra-fibrillar matrix was higher in the CEP (390kPa) compared to the NP (100kPa) or AF (30kPa). The permeability was very different across tissue regions, with the AF permeability (64 E−16m4/Ns) higher than the NP and CEP (~5.5 E−16m4/Ns). Additionally, a normalized time-constant (3000s) for the stress relaxation was similar for all the disc tissues. The properties measured in this study are important as benchmarks for tissue engineering and for modeling the disc's mechanical behavior and transport.