Human Lumbar Intervertebral Disc Research Articles

48. Bacterial presence in lumbar discs–subclinical infection or contamination? Insights from metabolomics

<h3>BACKGROUND CONTEXT</h3> The accumulating evidence associating subclinical infection with degenerative disc disease (DDD) and the controversy of contamination vs infection mandates further understanding of the microbial activity in the disc and host-microbiome interaction. <h3>PURPOSE</h3> To utilize a novel approach of metabolomics to probe the presence of bacterial metabolites involved in colonization, survival and replication in human lumbar intervertebral discs (LIVD). <h3>STUDY DESIGN/SETTING</h3> An observational case-control study. <h3>PATIENT SAMPLE</h3> Nucleus pulposus from the LIVD of three brain-dead voluntary organ donors (MRI normal and classified as controls) and of three patients undergoing surgery for degenerative disc disease (DDD) (cases) were utilized. <h3>OUTCOME MEASURES</h3> To prove presence of live and metabolically active bacteria in intervertebral disc. <h3>METHODS</h3> Untargeted metabolite profiling was carried out in six discs (3 controls and 3 cases) after extraction using methanol: acetonitrile: water (2 2:1) solvent system and acquired through HPLC-MS/MS platform using C18 reversed-phase column. From the total IVD metabolome, microbial metabolites were filtered by mapping against HMDB, ChEBI, SigMol, Siderophore database, ecdmb database, and PaMet databases. Biological functions of the metabolites were then studied by MSEA pipeline from Metaboanalyst, and the enrichment ratio, p-value, and Variably Importance Projection scores (VIPS) of the metabolites calculated. Degeneration responsive changes in the abundance of the microbial metabolites were calculated based on the peak intensities between the control and cases. <h3>RESULTS</h3> Mass spectrometry identified a total of 17601 and 15003 metabolites, respectively, in the control and diseased discs. Preliminary mapping of the above metabolites against HMDB indicated the multiple sources and of these, 64 metabolites were of microbial origin, accounting for 1.6% of the total IVD metabolome. Principle Component Analysis (PCA) and Orthogonal Partial Least Square-Discriminant Analysis (OPLSD-DA) showed distinct clustered patterns between control and disease, indicating a strong variation in concentration, peak and spectral values of the 64 metabolites between controls and cases. After exclusion of metabolites that were also associated with humans, drugs and food, 39 metabolites specific to bacteria were isolated. Nine were primary related to bacterial growth and survival, and the remaining 30 were secondary related to different environmental stress response activities. The three significant pathways (p 1.0) was found for nine metabolites which included (S)-14-methyilhexadecanoic acid related to P.acnes, 9-OxoODE, and 13-OxoODE related to gut flora, vibriobactin - a siderophore, tuberculosinol and iso-tuberculosinol, virulence factors of M.tuberculosis. There was also upregulation of autoinducer- 2, an important "Quorum sensing molecule" involved in bacterial cross-talk. <h3>CONCLUSIONS</h3> We identified several bacterial-specific metabolites participating in pathways involved in bacterial growth, survival and cross-talk. These were found in both groups but upregulated in diseased discs. Presence of quorum sensing molecules and cell-cell interactions provides firm proof of colonization and growth. These findings indicate that bacterial presence is not mere contamination but a colonization with possible role in infection-mediated inflammaging in DDD. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Smad7 Is Highly Expressed in Human Degenerative Discs and Participates in IL-1β-Induced Apoptosis of Rat AF Cells via the Mitochondria Pathway.

Background Abnormal Smad7 expression can lead to apoptosis in different cell types. Previously, we found high expression of Smad7 in rat degenerative discs. However, the exact role of Smad7 in the apoptosis of disc cells and the possible underlying mechanism remain unclear. Methods Degenerative and nondegenerative human lumbar intervertebral discs were collected from patients during operation. The expressions of SMAD7 mRNA and protein in the different components of these discs were measured with real-time PCR and Western blotting, respectively. Annulus fibrosus (AF) cells were isolated and cultivated from the discs of young healthy rats. Smad7 in the AF cells was overexpressed with adenovirus and knocked down with siRNA. IL-1β was used to induce apoptosis in the AF cells. Loss-and-gain cell function experiments were performed to show the effect of Smad7 on the apoptosis of AF cells. The function recovery experiments were performed to verify whether Smad7 regulates the apoptosis of AF cells through the mitochondria-mediated pathway. Results Both the mRNA and protein expressions of Smad7 were significantly higher in the different components of human degenerative discs than in those of the nondegenerative discs. IL-1β stimulated apoptosis while upregulating the Smad7 expression in the AF cells in vitro. Overexpression of Smad7 in AF cells exaggerated the IL-1β-induced apoptosis in the cells while knockdown of Smad7 expression suppressed this apoptosis. With the exaggerated apoptosis in the AF cells with Smad7 overexpression, both active cleaved caspase-3 and cleaved caspase-9, the ratio of Bax/Bcl-2, and Cyt-c increased significantly. However, the inhibitor of caspase-9, Z-LEHD-FMK, significantly diminished the apoptosis in these cells. Conclusion Smad7 is highly expressed in human degenerative discs and participates in IL-1β-induced apoptosis of rat AF cells via the mitochondria pathway. Smad7 may be a potential target for the prevention and treatment of degenerative disc disease.

Bacteria in human lumbar discs – subclinical infection or contamination? Metabolomic evidence for colonization, multiplication, and cell-cell cross-talk of bacteria

The accumulating evidence associating sub-clinical infection with disc degeneration (DD) and the controversy of contamination versus infection mandates a further understanding of the microbial activity in the disc and host-microbiome interaction. To utilize a novel approach of metabolomics to probe the presence of bacterial metabolites involved in colonization, survival, and replication in human lumbar intervertebral discs (LIVD). An observational case-control study. Nucleus pulposus from the LIVD of three brain-dead voluntary organ donors (MRI normal and classified as controls) and of three patients undergoing surgery for disc degeneration (DD) (cases) were utilized. Untargeted metabolite profiling was carried out in six discs (3-controls and 3-cases) after extraction using methanol: acetonitrile: water (2:2:1) solvent system and acquired through HPLC-MS/MS platform using C18 reversed-phase column. From the total IVD metabolome, microbial metabolites were filtered by mapping against HMDB, ChEBI, SigMol, Siderophore database, ecdmb database, and PaMet databases. The biological functions of the metabolites were then studied by MSEA pipeline from Metaboanalyst, and the enrichment ratio, p-value, and Variably Importance Projection scores of the metabolites were calculated. Degeneration responsive changes in the abundance of the microbial metabolites were calculated based on the peak intensities between the control and cases. Mass spectrometry identified a total of 17601 and 15003 metabolites, respectively, in the control and degenerated discs. Preliminary mapping of the above metabolites against HMDB indicated the multiple sources, and of these, 64 metabolites were of microbial origin, accounting for 1.6% of the total IVD metabolome. Principle Component Analysis and Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA) showed distinct clustered patterns between control and disc degene`ration, indicating a strong variation in concentration, peak, and spectral values of the 64 metabolites between controls and cases. After the exclusion of metabolites that were also associated with humans, drugs, and food, 39 metabolites specific to bacteria were isolated. Nine were primary metabolites related to bacterial growth and survival, and the remaining 30 were secondary metabolites related to different environmental stress response activities. The three significant pathways (p<.001) which were predominant in the bacterial metabolites were autoinducer-2 biosynthesis, peptidoglycan biosynthesis, and chorismate pathway. In addition, a significant fold change of >1.0 was found for nine metabolites which included (S)-14-Methyilhexadecanoic acid related to P. acnes, 9-OxoODE, and 13-OxoODE related to gut flora, vibriobactin - a siderophore, tuberculosinol and iso-tuberculosinol, virulence factors of M. tuberculosis. There was also upregulation of Autoinducer- 2, an important "Quorum sensing molecule" involved in bacterial cross-talk. We identified several bacterial-specific metabolites participating in bacterial growth, survival, and cross-talk pathways. These were found in both groups but up-regulated in degenerated discs. The presence of Quorum sensing molecules and cell-cell interactions provides firm proof of colonization and growth. These findings indicate that the bacterial presence may not be mere contamination but could be colonization with a possible role in infection-mediated inflammation in DD. Proof of subclinical infection as an initiator of DD and documentation of exact germ and drug sensitivity will change the way millions of patients with non-specific low back pain (NSLBP) are treated across the world.

DNA Methylation in Degenerating Human Intervertebral Discs

Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP) and is associated with widespread changes in gene expression, extracellular matrix (ECM) degradation, inflammation, and innervation. Epigenetic modifications, including DNA methylation, can drive changes in gene expression without modifying DNA sequences. To date, little is known about epigenetic dysregulation in degenerated IVDs. The objective of this study was to investigate changes in DNA methylation in degenerating human IVDs from chronic LBP patients. Human lumbar IVD segments at L4/5 level including control (n=4) and degenerating (n=6) were homogenized in liquid nitrogen with a mortar and pestle and genomic DNA was extracted using the Qiagen DNeasy Blood and Tissue kit. Epigenome-wide 5mC mapping was performed following bisulfite sequencing using the Infinium MethylationEPIC BeadChip 850k Arrays. All analysis was performed in the R package RnBeads 2.0. Principal component analysis and hierarchical clustering analysis revealed group differences between healthy and degenerating IVDs. Pathway analysis revealed enrichment in genes related to ECM organization, signaling by receptor tyrosine kinases and signaling by interleukins. Further analysis at the single gene promoter level showed hypermethylation in ECM-related genes linked to IVD degeneration including sparc and col2a1 and hypomethylation of pro-inflammatory cytokines associated with IVD degeneration including IL-1b and IL-8. These changes in methylation would be expected to result in loss of ECM and increased inflammation. Persistent reprogramming of gene expression by DNA methylation could drive IVD degeneration and inflammation. Understanding the role of DNA methylation in IVD pathology may provide a scientific framework for the development of new therapeutic approaches.

Can TRIF/TICAM-1 Dependent Pathway be Target Pathway in Lumbar Intervertebral Disc Degeneration?

The current study aims to elucidate the role of the TIR-domain-containing adaptor-inducing interferon-β (TRIF) dependent pathway in intervertebral disc degeneration (IVD). 88 adult male patients with low back pain (LBP) (+/- radicular pain) were further evaluated by magnetic resonance imaging (MRI) with surgical indication for microscopic lumbar disc herniation (LDH). Preoperatively, patients were classified according to Modic Changes (MC), use of nonsteroidal anti-inflammatory drugs (NSAIDs), and the presence of radicular pain additional to the LBP. The age of the 88 patients ranged from 19 to 75 years (mean: 47.3 ± 19.6 years). 28 of the patients were evaluated as MC I (31.8%), 40 as MC II (45.4%), and 20 as MC III (22.7%). The majority of patients (81.8%) had radicular LBP, while 16 patients (18.1%) had only LBP. Predominantly, 55.6% of all patients were taking NSAIDs. Levels of all adaptor molecules were highest in the MC I group and lowest in the MC III group. The levels of IRF3, TICAM1, TICAM2, NF-kB p65, TRAF6, and TLR4 were significantly increased in the MC I group compared to the MC II and MC III groups. The variations of the individual adaptor molecules showed no statistically significant difference in the use of NSAIDs and radicular LBP. As a result of the impact assessment, the current study clearly demonstrated for the first time that the TRIF-dependent signalling pathway plays a crucial role in the degeneration process in human lumbar intervertebral disc specimens.

A microstructure-based model for a full lamellar-interlamellar displacement and shear strain mapping inside human intervertebral disc core

The determinant role of the annulus fibrosus interlamellar zones in the intervertebral disc transversal and volumetric responses and hence on their corresponding three-dimensional conducts have been only revealed and appreciated recently. Their consideration in disc modeling strategies has been proven to be essential for the reproduction of correct local strain and displacement fields inside the disc especially in the unconstrained directions of the disc. In addition, these zones are known to be the starting areas of annulus fibrosus circumferential tears and disc delamination failure mode, which is often judged as one of the most dangerous disc failure modes that could evolve with time leading to disc hernia. For this latter reason, the main goal of the current contribution is to incorporate physically for the first time, the interlamellar zones, at the scale of a complete human lumbar intervertebral disc, in order to allow a correct local vision and replication of the different lamellar-interlamellar interactions and an identification of the interlamellar critical zones. By means of a fully tridimensional chemo-viscoelastic constitutive model, which we implemented into a finite element code, the physical, mechanical and chemical contribution of the interlamellar zones is added to the disc. The chemical-induced volumetric response is accounted by the model for both the interlamellar zones and the lamellae using experimentally-based fluid kinetics. Computational simulations are performed and critically discussed upon different simple and complex physiological movements. The disc core and the interlamellar zones are numerically accessed, allowing the observation of the displacement and shear strain fields that are compared to direct MRI experiments from the literature. Important conclusions about the correct lamellar-interlamellar-nucleus interactions are provided thanks to the developed model. The critical interlamellar spots with the highest delamination potentials are defined, analyzed and related to the local kinetics and microstructure.

Application of Magnetic Resonance-Ultra Time Echo (MR-UTE) Imaging in the Analysis of the Degree of Degeneration of the Intervertebral Disc Cartilage Endplate

Magnetic resonance imaging (MRI) is the most widely used imaging method in clinical lumbar spine examination. Because of its advantages of non-radiation and good tissue contrast, magnetic resonance imaging provides rich and effective diagnostic information for clinic. The most commonly used sequence is type 2 (T2) sequence, which has a longer time (usually longer than 2000 ms). It shows well in long T2 tissues such as nucleus pulposus, cerebrospinal fluid and adipose tissue, showing moderator high signal in images, while for short T2 tissues such as cartilage endplate and anterior and posterior longitudinal zone, it is often no signal and low signal because of its short attenuation time, thus forming obvious tissue contrast. But at the same time, because the time is too long, for short T2 tissue, the signal has been attenuated to zero before sequence acquisition, so the complete structure can not be displayed directly. In this paper, the normal human lumbar intervertebral disc was studied by conventional magnetic resonance type 1 (T1), T2 and double-echo-UTE imaging techniques. Each part of lumbar intervertebral disc and the semi-quantitative analysis of anatomical structure in images were compared, and the advantages and characteristics of each sequence for each anatomical structure of lumbar intervertebral disc and the advantage of MR-UTE in intervertebral disc display were discussed. It has been found that UTE, as a new sequence which can effectively image short T2 tissue, is gradually applied from experiment to clinic in bone and joint system because of its shorter time. In the gross specimens of lumbar intervertebral disc, sequence can directly display the cartilage endplate and the short T2 tissue of the anterior and posterior longitudinal ligament.

The Verification of Human Lumbar Vertebrae and Intervertebral Disc Finite Element Models under Mechanical Forces

Bone, being nonhomogeneous in nature need a complicated and time-consuming process to undergo computed simulation like finite element analysis. To overcome this hurdle, assuming a nonhomogeneous model as homogeneous could be a solution. The objective of this study is to focus on developing a homogeneous human lumbar finite element models and verify them under mechanical force by measuring disc stress, disc strain, disc deformation, total strain, and total deformation. Experimental and geometrical analysis were performed before verifying the lumbar model. To verify the models’ reliability, nonhomogeneous lumbar models were also developed. Five different static structural simulations were performed on four lumbar segments, and twenty parameters were measured. Numerically, out of twenty, eighteen parameters showed very less or no significant difference between homogeneous and nonhomogeneous models of the intervertebral discs and lumbar vertebrae. At the same time, proper caution to be provided while examining the results. With this validation procedure, researchers can process artifact images to get more information which enables them to contribute to the patient’s well-being.

Neuropeptide Y prevents nucleus pulposus cells from cell apoptosis and IL‑1β‑induced extracellular matrix degradation

ABSTRACT Intervertebral disc degeneration (IDD) is characterized by excessive inflammatory reaction, and neuropeptide Y (NPY) was reported to have anti-inflammatory effect. However, the effect of NPY on NP cells has not been investigated up to date. This study aimed to clarify the role of NPY on the process of IDD. Fourteen fresh human lumbar intervertebral discs were harvested, and degeneration-related proteins were examined. Pfirrmann grading system was used to evaluate IDD. Rat nucleus pulposus (NP) cells were used to investigate the effect of NPY on the proliferation, apoptosis, and extracellular matrix (ECM) in NP cell induced by IL-1βin vitro. The expression levels of NPY and its receptors (type 1 receptor, Y1R, and type 2 receptor, Y2R) were detected via immunohistochemical analysis, western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and proliferation were explored using cell counting kit-8 assay, western blot, and immunofluorescence analysis. Cell apoptosis was investigated by Hoechst staining, JC-1 Staining, annexin V-FITC/PI double staining, and western blot. The secretion of NPY from NP cells was determined via enzyme-linked immunosorbent assay (ELISA). The expression of anabolic and catabolic gene was analyzed by qRT-PCR, western blot, immunofluorescence analysis, and ELISA. The expression of Y2R was significantly increased in both human degenerative intervertebral discs and IL-1β-induced NP cells. Although no positive results for NPY indicated by western blot both in vivo and in vitro, ELISA results demonstrated that the secretion of NPY from NP cells was increased by low-concentration IL-1β, but was decreased when the concentration of IL-1β was 30 ng/ml and above. In addition, NPY could promote NP cells proliferation and protect NP cells against IL‑1β‑induced apoptosis via suppressing mitochondrial-mediated apoptosis pathway. What’s more, NPY can suppress the expression of catabolic gene and ameliorate IL-1β- induced matrix degeneration in NP cells. In conclusion, NPY could promote NP cell proliferation and alleviate IL‑1β‑induced cell apoptosis via mitochondrial pathway. In addition, NPY can suppress the expression of ECM‑catabolic proteinases and ameliorate IL-1β- induced ECM degeneration in vitro.

Damage to the human lumbar cartilage endplate and its clinical implications.

The cartilaginous endplate (CEP) is a thin layer of hyaline cartilage, and plays an important role in the diffusion of nutrients into the intervertebral discs. Its damage may seriously affect the disc degeneration, and result in low back pain (LBP). However, the structural features of damaged CEPs have not been well characterized, and this hinders our understanding of the etiology of disc degeneration and pain. To present the structural features of micro-damaged CEPs in patients with disc degeneration and LBP that might even be regarded as an initial factor for disc degeneration, we performed a histological study of micro-damaged CEPs harvested from human lumbar intervertebral discs and analyzed its clinical implications. Human lumbar CEPs were excised from 35 patients (mean age 60.91years) who had disc degeneration and LBP. Control tissue was obtained from 15 patients (mean age 54.67years) with lumbar vertebral burst fractures. LBP and disability were assessed clinically, and all patients underwent anterior vertebral body fusion surgery. CEPs together with some adjacent nucleus pulposus (NP) were sectioned at 4µm, and stained using H&E, Safranin O/Fast Green, and Alcian Blue. Immunostaining and PCR were used to identify various markers of degeneration, innervation, and inflammation. Histology demonstrated physical micro-damage in 14/35 CEPs from the disc degeneration group. Six major types of damage could be distinguished: fissure, traumatic nodes, vascular mimicry, incorporation of NP tissue within the CEP, incorporation of bone within the CEP, and incorporation of NP and bone within the CEP. Pain and disability scores (ODI: p=0.0190; JOA: p=0.0205; JOABPEQ: p=0.0034) were significantly higher in those with micro-damaged CEPs (N=14) than in those with non-damaged CEPs (N=21). CEP damage was significantly associated with elevated MMP3 (p=0.043), MMP13 (p=0.0191), ADAMTS5 (p=0.0253), TNF-α (p=0.0011), and Substance P (p=0.0028), and with reduced Sox9 (p=0.0212), aggrecan (p=0.0127), and type II collagen (p=0.0139). In conclusion, we presented a new classification of human lumbar micro-damaged CEPs. Furthermore, we verify disc degeneration, innervation, and discogenic pain in micro-damaged CEPs.

Effect of intervertebral disc degeneration on mechanical and electric signals at the interface between disc and vertebra

Intervertebral disc (IVD) degeneration is significantly correlated with the changes in structure and material properties of adjacent vertebral bone, possibly through mechanical and electrical interactions. However, the mechanisms underlying the alteration of the mechanical and electrical environment at the disc-vertebra interface related with disc degeneration have not been well studied. The objective of this study was to numerically investigate the long-term distributions of mechanical and electrical signals on the disc-vertebra interface with disc degeneration. A three-dimensional finite element model of a human lumbar IVD was used to study the mechanical and electric signals at the interface between disc and vertebral body. The disc degeneration was simulated by reducing the nutrition levels on the nucleus pulposus (NP)-vertebra interface and on the annulus fibrosus (AF) periphery to 30% and 60% of its normal values, respectively. In the simulation, the total external mechanical load applied to the disc-vertebra segment was assumed unchanged during disc degeneration. The simulation results showed that the compressive stress of solid matrix changed by up to ~37 kPa on the NP-vertebra interface, while it increased by up to ~32 kPa on the AF-vertebra interface. The shear stress increased by up to ~37 kPa with disc degeneration. The absolute value of the electric potential on the disc-vertebra interface of the disc slightly decreased with the disc degeneration (~0.5 mV). The knowledge of these spatial and temporal variations of the mechanical stresses and electric potential on the disc-vertebra interface is important for understanding the vertebrae adaptation and remodeling during disc degeneration.

Quantifying deformations and strains in human intervertebral discs using Digital Volume Correlation combined with MRI (DVC-MRI)

Physical disruptions to intervertebral discs (IVDs) can cause mechanical changes that lead to degeneration and to low back pain which affects 75% of us in our lifetimes. Quantifying the effects of these changes on internal IVD strains may lead to better preventative strategies and treatments. Digital Volume Correlation (DVC) is a non-invasive technique that divides volumetric images into subsets, and measures strains by tracking the internal patterns within them under load. Applying DVC to MRIs may allow non-invasive strain measurements. However, DVC-MRI for strain measurements in IVDs has not been used previously. The purpose of this study was to quantify the strain and deformation errors associated with DVC-MRI for measurements in human IVDs.Eight human lumbar IVDs were MRI scanned (9.4 T) for a ‘zero-strain study’ (multiple unloaded scans to quantify noise within the system), and a loaded study (2 mm axial compression). Three DVC methodologies: Fast-Fourier transform (FFT), direct correlation (DC), and a combination of both FFT and DC approaches were compared with subset sizes ranging from 8 to 88 voxels to establish the optimal DVC methodology and settings which were then used in the loaded study.FFT + DC was the optimal method and a subset size of 56 voxels (2520 µm) was found to be a good compromise between errors and spatial resolution. Displacement and strain errors did not exceed 28 µm and 3000 microstrain, respectively.These findings demonstrate that DVC-MRI can quantify internal strains within IVDs non-invasively and accurately. The method has unique potential for assessing IVD strains within patients.

Rapid determination of internal strains in soft tissues using an experimentally calibrated finite element model derived from magnetic resonance imaging.

Finite element models (FEMs) of medical images can provide information about the underlying tissue that cannot be obtained from the original images. Preforming an accurate simulation requires the careful experimental calibration of boundary conditions. Here we describe a method for deriving a geometric mesh for soft biological materials using a magnetic resonance imaging (MRI) system, and an experimental workflow for calibrating the boundary conditions and optimizing the mesh density in these simulations. A three-dimensional image stack of a ballistic sphere gel, a bovine caudal intervertebral disc (IVD), and a human lumbar IVD were generated using a positional MRI system. These images were then segmented using a semi-automated process, converted to a tetrahedral mesh, and then modeled as a linear elastic solid. The mesh density was optimized based on simulation time and convergence with the experimental results. The modulus of the ballistic gel was determined experimentally, while the material properties for the nucleus pulposus (NP) and the annulus fibrosus (AF) within the bovine and human IVDs were assigned from literature. The simulation for the spherical gel and the bovine IVD matched the reaction forces determined experimentally in compression. We then simulated a 0.3 MPa compressive load on the human lumbar IVD at the optimal mesh density and material properties determined from the bovine model and then examined the resultant internal strains. The scaled mesh density demonstrated excellent correspondence with the experimental results, confirming that accuracy was not compromised. Both the ballistic gel and the IVD samples exhibited a wide range of internal strains. The NP of the IVD underwent greater deformation than the AF under loading. This study validated a strategy for mesh optimization and FEM of soft biological materials from data generated from MRI scans. This calibrated approach allows for the rapid examination of internal strain distributions medical images that can be performed on the order of minutes.

Fracture strength estimation of L3-L4 intervertebral disc using FEA

Mechanical stress and fracture analysis of the human lumbar intervertebral discs are important in assessing disorders related to lower back pain and ageing. Finite element modelling and simulation approaches assist in easier prediction of the disc behaviour under different load conditions. The causes of mechanical failure and morphological changes still remain partially speculative. The present study addresses the issue by developing a finite element model of an L3-L4 lumbar intervertebral disc subjected to different axial compressive loadings. The morphological deformations and stress concentration regions within the disc are analyzed and reported. A mathematical relation is established to estimate the breaking strength of an L3-L4 intervertebral disc, thus indicating the risk of disc failure based on the applied load.

Reciprocal Regulation of TRPS1 and miR-221 in Intervertebral Disc Cells.

Intervertebral disc (IVD), a moderately moving joint located between the vertebrae, has a limited capacity for self-repair, and treating injured intervertebral discs remains a major challenge. The development of innovative therapies to reverse IVD degeneration relies primarily on the discovery of key molecules that, occupying critical points of regulatory mechanisms, can be proposed as potential intradiscal injectable biological agents. This study aimed to elucidate the underlying mechanism of the reciprocal regulation of two genes differently involved in IVD homeostasis, the miR-221 microRNA and the TRPS1 transcription factor. Human lumbar IVD tissue samples and IVD primary cells were used to specifically evaluate gene expression and perform functional analysis including the luciferase gene reporter assay, chromatin immunoprecipitation, cell transfection with hTRPS1 overexpression vector and antagomiR-221. A high-level expression of TRPS1 was significantly associated with a lower pathological stage, and TRPS1 overexpression strongly decreased miR-221 expression, while increasing the chondrogenic phenotype and markers of antioxidant defense and stemness. Additionally, TRPS1 was able to repress miR-221 expression by associating with its promoter and miR-221 negatively control TRPS1 expression by targeting the TRPS1-3′UTR gene. As a whole, these results suggest that, in IVD cells, a double-negative feedback loop between a potent chondrogenic differentiation suppressor (miR-221) and a regulator of axial skeleton development (TRPS1) exists. Our hypothesis is that the hostile degenerated IVD microenvironment may be counteracted by regenerative/reparative strategies aimed at maintaining or stimulating high levels of TRPS1 expression through inhibition of one of its negative regulators such as miR-221.

Material properties of human lumbar intervertebral discs across strain rates

BACKGROUND CONTEXTThe use of finite element (FE) methods to study the biomechanics of the intervertebral disc (IVD) has increased over recent decades due to their ability to quantify internal stresses and strains throughout the tissue. Their accuracy is dependent upon realistic, strain-rate dependent material properties, which are challenging to acquire. PURPOSEThe aim of this study was to use the inverse FE technique to characterize the material properties of human lumbar IVDs across strain rates. STUDY DESIGNA human cadaveric experimental study coupled with an inverse finite element study. METHODSTo predict the structural response of the IVD accurately, the material response of the constituent structures was required. Therefore, compressive experiments were conducted on 16 lumbar IVDs (39±19 years) to obtain the structural response. An FE model of each of these experiments was developed and then run through an inverse FE algorithm to obtain subject-specific constituent material properties, such that the structural response was accurate. RESULTSExperimentally, a log-linear relationship between IVD stiffness and strain rate was observed. The material properties obtained through the subject-specific inverse FE optimization of the annulus fibrosus (AF) fiber and AF fiber ground matrix allowed a good match between the experimental and FE response. This resulted in a Young modulus of AF fibers (—MPa) to strain rate (ε˙, /s) relationship of YMAF=31.5ln(ε˙)+435.5, and the C10 parameter of the Neo-Hookean material model of the AF ground matrix was found to be strain-rate independent with an average value of 0.68 MPa. CONCLUSIONSThese material properties can be used to improve the accuracy, and therefore predictive ability of FE models of the spine that are used in a wide range of research areas and clinical applications. CLINICAL SIGNIFICANCEFinite element models can be used for many applications including investigating low back pain, spinal deformities, injury biomechanics, implant design, design of protective systems, and degenerative disc disease. The accurate material properties obtained in this study will improve the predictive ability, and therefore clinical significance of these models.

Morphological characteristics of the kangaroo lumbar intervertebral discs and comparison with other animal models used in spine research.

Animal models are frequently used to elucidate pathomechanism and pathophysiology of various disorders of the human intervertebral disc (IVD) and also to develop therapeutic approaches. Here we report morphological characteristics of the kangaroo lumbar IVDs and compare them with other animal models used in spine research. Twenty-five fresh-frozen cadaveric lumbar spines (T12-S1) derived from kangaroo carcases (Macropus giganteus) of undetermined age were first scanned in a C-Arm X-ray machine. A photograph of the axial section of the disc including a calibrated metric scale was also acquired. The digital radiographs and photographs were processed in ImageJ to determine the axial and sagittal plane dimensions for the whole disc (WD) and the nucleus pulposus (NP) and the mid-sagittal disc height for all the lumbar levels. Our results suggest that the L6-S1 IVD in kangaroos is distinctly large compared with the upper lumbar IVDs. Based on previously published data, human lumbar IVDs are the largest of all the animal IVDs used in spine research, with camelid cervical IVDs being the closest relative in absolute dimensions (llamas: 78% in disc height, 40% in WD volume, and 38% in NP volume). Kangaroo L6-S1 IVD was approximately 51% in height, 20% in WD volume, and 20% in NP volume of the human lumbar IVD. We conclude that morphological similarities exist between a kangaroo and human lumbar IVD, especially with the lima bean shape in the axial plane, wedge shape in the sagittal plane, convexity at the cephalad endplates, and percentage volume occupied by the NP in the IVD. These slides can be retrieved under Electronic Supplementary Material.

Normal aging in human lumbar discs: An ultrastructural comparison.

The normal aging of the extracellular matrix and collagen content of the human lumbar intervertebral disc (IVD) remains relatively unknown despite vast amounts of basic science research, partly because of the use of inadequate surrogates for a truly normal, human IVD. Our objective in this study was to describe and compare the morphology and ultrastructure of lumbar IVDs in 2 groups of young (G1—<35 years) and elderly (G2—>65 years). Thirty L4-5 and L5-S1 discs per group were obtained during autopsies of presumably-asymptomatic individuals and analyzed with magnetic resonance imaging (MRI), a morphological grading scale, light microscopy, scanning electron microscopy (SEM) and immunohistochemistry (IHC) for collagen types I, II, III, IV, V, VI, IX and X. As expected, a mild to moderate degree of degeneration was present in G1 discs and significantly more advanced in G2. The extracellular matrix of G2 discs was significantly more compact with an increase of cartilaginous features such as large chondrocyte clusters. Elastic fibers were abundant in G1 specimens and their presence correlated more with age than with degeneration grade, being very rare in G2. SEM demonstrated persistence of basic structural characteristics such as denser lamellae with Sharpey-type insertions into the endplates despite advanced age or degeneration grades. Immunohistochemistry revealed type II collagen to be the most abundant type followed by collagen IV. All collagen types were detected in every disc sector except for type X collagen. Statistical analysis demonstrated a general decrease in collagen expression from G1 to G2 with an annular- and another nuclear-specific pattern. These results suggest modifications of IVD morphology do not differ between the anterior or posterior annulus but are more advanced or happen earlier in the posterior areas of the disc. This study finally describes the process of extracellular matrix modification during disc degeneration in an unselected, general population and demonstrates it is similar to the same process in the cervical spine as published previously.

Thermoreversible hyaluronan-hydrogel and autologous nucleus pulposus cell delivery regenerates human intervertebral discs in an ex vivo, physiological organ culture model.

Numerous studies show promise for cell-based tissue engineering strategies aiming to repair painful intervertebral disc (IVD) degeneration. However, clinical translation to human IVD repair is slow. In the present study, the regenerative potential of an autologous nucleus pulposus (NP)-cell-seeded thermoresponsive hyaluronic acid hydrogel in human lumbar IVDs was assessed under physiological conditions. First, agarose-encased in vitro constructs were developed, showing greater than 90 % NP cell viability and high proteoglycan deposition within HA-pNIPAM hydrogels following 3 weeks of dynamic loading. Second, a bovine-induced IVD degeneration model was used to optimise and validate T1ρ magnetic resonance imaging (MRI) for detection of changes in proteoglycan content in isolated intact IVDs. Finally, isolated intact human lumbar IVDs were pre-scanned using the established MRI sequence. Then, IVDs were injected with HA-pNIPAM hydrogel alone or autologous NP-cell-seeded. Next, the treated IVDs were cultured under cyclic dynamic loading for 5 weeks. Post-treatment T1ρ values were significantly higher as compared to pre-treatment scans within the same IVD and region of interest. Histological evaluation of treated human IVDs showed that the implanted hydrogel alone accumulated proteoglycans, while those that contained NP cells also displayed neo-matrix-surrounded cells within the gel. The study indicated a clinical potential for repairing early degenerative human IVDs using autologous cells/hydrogel suspensions. This unique IVD culture set-up, combined with the long-term physiological culture of intact human IVDs, allowed for a more clinically relevant evaluation of human tissue repair and regeneration, which otherwise could not be replicated using the available in vitro and in vivo models.

Changes in human intervertebral disc biochemical composition and bony end plates between middle and old age.

ObjectiveThis study evaluates molecular, nutritional and biochemical alterations in human intervertebral discs between middle and old age.MethodsTwenty-eight human lumbar intervertebral discs from donors were evaluated and separated into two groups: Middle-aged (35–50 years old, relatively non-degenerate discs of Pfirrmann grades 1–3, n = 15) and Old-aged (≥80 years old, all degenerate Pfirrmann grade 4 or 5, n = 13). Parameters which might be expected to to be related to nutrient supply and so the health of disc cells (eg the porosity of the vertebral endplate, cell viability and cell density) and to disc extracellular composition (ie quantification of glycosaminoglycan disaccharides and hyaluronic acid molecular weight) and collagen organization, were analyzed. Three regions of the intervertebral disc (anterior annulus fibrosus, nucleus pulposus, and posterior annulus fibrosus) were examined.ResultsThe old-aged group showed a decrease in content of sulphated and non-sulphated glycosaminoglycans relative to middle-aged and there were also alterations in the proportion of GAG disaccharides and a decrease of collagen fiber size. Hyaluronic acid molecular weight was around 200 kDa in all regions and ages studied. The anterior annulus differed from the posterior annulus particularly in relation to cell density and GAG content. Additionally, there were changes in the bony endplate, with fewer openings observed in the caudal than cranial endplates of all discs in both groups.ConclusionsResults show the cranial vertebral endplate is the main vascular source for the intervertebral discs. Hylauronic acid molecular weight is the same through the intervertebral disc after age of 50 years.