Aims. This study aimed, through bioinformatics analysis and in vitro experiment validation, to identify the key extracellular proteins of
Aims. Interleukin (IL)-1β is one of the major pathogenic regulators during the pathological development of
Aims. In this investigation, we administered oxidative stress to nucleus pulposus cells (NPCs), recognized DNA-damage-inducible transcript 4 (DDIT4) as a component in
Objectives. Interleukin 18 (IL-18) is a regulatory cytokine that degrades the disc matrix. Bone morphogenetic protein-2 (BMP-2) stimulates synthesis of the disc extracellular matrix. However, the combined effects of BMP-2 and IL-18 on human
Using deep learning and image processing technology, a standardized automatic quantitative analysis systerm of lumbar disc degeneration based on T2MRI is proposed to help doctors evaluate the prognosis of
Purpose of study and background. Clinical researchers use Pfirrmann classification for grading
Current therapies for
It has been proposed that
Biomechanical overloading initiates
Introduction: Osteoporotic fractures affect certain bones more than others, suggesting that systemic bone loss is not the only underlying cause. We have shown that age-related
Low back pain affects 80% of the population with half of cases attributed to
Background. The association between lumbar
Purpose of study and background. Mechanical overloading initiates
Back pain is a leading cause of disability worldwide and it is primarily considered to be triggered by
Introduction.
Mesenchymal stem cells (MSCs) are exciting candidates for cellular repopulation and repair in
Purpose of study and background. Low back pain affects 80% of the population at some point in their lives with 40% of cases attributed to
Background.
Background. Endplate defect is an MRI trait, found to be associated with
Degenerative disc disease (DDD) is a common cause of lower back pain. Calcification of the intervertebral disc (IVD) has been correlated with DDD, and is especially prevalent in scoliotic discs. The appearance of calcium deposits has been shown to increase with age, and its occurrence has been associated with several other disorders such as hyperparathyroidism, chondrocalcinosis, and arthritis. Trauma, vertebral fusion and infection have also been shown to increase the incidence of IVD calcification. Our data indicate that Ca2+ and expression of the extracellular calcium-sensing receptor (CaSR) are significantly increased in mild to severely degenerative human IVDs. In this study, we evaluated the effects of Ca2+ and CaSR on the degeneration and calcification of IVDs. Human donor lumbar spines of Thompson grade 2, 3 and 4 through organ donations within 24 hs after death. IVD cells, NP and AF, were isolated from tissue by sequential digestion with Pronase followed by Collagenase. Cells were expanded for 7 days under standard cell culture conditions. Immunohistochemistry was performed on IVD tissue to validate the grade and expression of CaSR. Free calcium levels were also measured and compared between grades. Immunocytochemistry, Western blotting and RT-qPCR were performed on cultured NP and AF cells to demonstrate expression of CaSR, matrix proteins aggrecan and collagen, catabolic enzymes and calcification markers. IVD cells were cultured in increasing concentrations of Ca2+ [1.0-5.0 mM], CaSR allosteric agonist (cincalcet, 1 uM), and IL-1b [5 ng/mL] for 7 days. Ex vivo IVD organ cultures were prepared using PrimeGrowth Disc Isolation System (Wisent Bioproducts, Montreal, Quebec). IVDs were cultured in 1.0, 2.5 mM Ca2+ or with cinacalcet for 21 days to determine effects on disc degeneration, calcification and biomechanics. Complex modulus and structural stiffness of disc tissues was determined using the MACH-1 mechanical testing system (Biomomentum, Laval, Quebec). Ca2+ dose-dependently decreased matrix protein synthesis of proteoglycan and Col II in NP and AF cells, similar to treatment with IL-1b. (n = 4). Contrarily to IL-1b, Ca2+ and cincalcet did not significantly increase the expression of catabolic enzymes save ADAMTS5. Similar effects were observed in whole organ cultures, as Ca2+ and cinacalcet decreased proteoglycan and collagen content. Although both Ca2+ and cinacalcet increased the expression of alkaline phosphatase (ALP), only in Ca2+-treated IVDs was there evidence of calcium deposits in NP and AF tissues as determined by von Kossa staining. Biomechanical studies on Ca2+ and cinacalcet-treated IVDs demonstrated decreases in complex modulus (p<0.01 and p<0.001, respectively; n=5), however, only Ca2+-treated IVDs was there significant increases stiffness in NP and AF tissues (p<0.001 and p<0.05, respectively; n=3). Our results suggest that changes in the local concentrations of calcium and activation of CaSR affects matrix protein synthesis, calcification and IVD biomechanics. Ca2+ may be a contributing factor in IVD degeneration and calcification.
A key cause of low back pain is the degeneration of the intervertebral disc (IVD). Causality between infection of the IVD and its degenerative process gained great interest over the last decade. Granville Smith et al. (2021) identified 36 articles from 34 research studies investigating bacteria in human IVDs. Bacteria was identified in 27 studies, whereas 9 attributed bacterial presence to contamination. Immunohistochemical staining for Gram positive bacteria was performed on human IVD tissue to identify presence and characterise bacterial species. Nucleus pulposus (NP) cells in monolayer and 3D alginate were stimulated with LPS and Peptidoglycan (0.1-50 µg/ml) for 48hrs. Following stimulation qPCR for factors associated with disc degeneration including matrix genes, matrix degrading enzymes, cytokines, neurotrophic factors and angiogenic factors and conditioned media collected for ELISA and luminex analysis Gram positive bacteria was detected within human IVD tissue. Internalisation of bacteria by NP cells influenced the cell and nuclei morphology. Preliminary results of exposure of NP cells to bacterial components indicate that LPS as well as Peptidoglycan increase IL-8 and ADAMTS-4 gene expression following 48 hours of stimulation with a dose response seen for IL-8 induction by peptidoglycan compared to the control group. Underlining these results, IL-8 protein release was increased for treated groups compared to non-treated control. Further analysis is underway investigating other output measures and additional biological repeats. This study has demonstrated bacteria are present within IVD cells within IVD tissue removed from degenerate IVD and is determining the potential influence of these on disc degeneration.
Low back pain is strongly associated with degeneration of the intervertebral disc (IVD). During degeneration, altered matrix synthesis and increased matrix degradation, together with accompanied cell loss is seen particularly in the nucleus pulposus (NP). It has been proposed that notochordal (NC) cells, embryonic precursors for the cells within the NP, could be utilized for mediating IVD regeneration. However, injectable biomaterials are likely to be required to support their phenotype and viability within the degenerate IVD. Therefore, viability and phenotype of NC cells were analysed and compared within biomaterial carriers subjected to physiological oxygen conditions over a four-week period were investigated. Porcine NC cells were incorporated into three injectable hydrogels: NPgel (a L-pNIPAM-co-DMAc hydrogel), NPgel with decellularized NC-matrix powder (dNCM) and Albugel (an albumin/ hyaluronan hydrogel). The NCs and biomaterials constructs were cultured for up to four weeks under 5% oxygen (n=3 biological repeats). Histological, immunohistochemical and glycosaminoglycans (GAG) analysis were performed to investigate NC viability, phenotype and extracellular matrix synthesis and deposition.Objectives
Methodology
Low back pain is strongly associated with degeneration of the intervertebral disc (IVD). During degeneration, altered matrix synthesis and increased matrix degradation, together with accompanied cell loss is seen particularly in the nucleus pulposus (NP). It has been proposed that notochordal (NC) cells, embryonic precursors for the cells within the NP, could be utilized for mediating IVD regeneration. However, injectable biomaterials are likely to be required to support their phenotype and viability within the degenerate IVD. Therefore, viability and phenotype of NC cells were analysed and compared within biomaterial carriers subjected to physiological oxygen conditions over a four-week period were investigated. Porcine NC cells were incorporated into three injectable hydrogels: NPgel (a L-pNIPAM-co-DMAc hydrogel), NPgel with decellularized NC-matrix powder (dNCM) and Albugel (an albumin/ hyaluronan hydrogel). The NCs and biomaterials constructs were cultured for up to four weeks under 5% oxygen (n=3 biological repeats). Histological, immunohistochemical and glycosaminoglycans (GAG) analysis were performed to investigate NC viability, phenotype and extracellular matrix synthesis and deposition. Histological analysis revealed that NCs survive in the biomaterials after four weeks and maintained cell clustering in NPgel, Albugel and dNCM/NPgel with maintenance of morphology and low caspase 3 staining. NPgel and Albugel maintained NC cell markers (brachyury and cytokeratin 8/18/19) and extracellular matrix (collagen type II and aggrecan). Whilst Brachyury and Cytokeratin were decreased in dNCM/NPgel biomaterials, Aggrecan and Collagen type II was seen in acellular and NC containing dNCM/NPgel materials. NC containing constructs excreted more GAGs over the four weeks than the acellular controls. NC cells maintain their phenotype and characteristic features in vitro when encapsulated into biomaterials. NC cells and biomaterial construct could potentially become a therapy to treat and regenerate the IVD.
Purpose:
The purpose of this study was to investigate the possible relationship between matrix metalloproteinase-3 (MMP-3) promoter 5A/6A polymorphism and
Introduction:
Current medical treatments for IVD degeneration rely on conservative therapies or surgery. Surgical treatments (e.g. spinal fusion,) have shown satisfactory results in alleviating pain, but long-term clinical outcomes remain poor. Thus, there is an urgent need for alternative cell based regenerative therapies focussed on correcting the underlying pathogenesis of IVD degeneration. However, for these to be successful an appropriate cell source for implantation, together with a suitable growth factor to direct cell differentiation and formation of a functional matrix must be identified. Additionally, extensive
Injectable hydrogels via minimally invasive surgery reduce the risk of infection, scar formation and the cost of treatment. Degradation of the intervertebral disc (IVD) currently has no preventative treatment. An injectable hydrogel material could restore disc height, reinforce local mechanical properties, and promote tissue regeneration. We present a hydrogel material Laponite® associated poly(N-isopropylacrylamide)-co-poly(dimethylacrylamide) (NPGel). Understanding how the components of this hydrogel system influence material properties, is crucial for tailoring treatment strategies for the IVD and other tissues. The effect of hydrogel wt./wt., clay and co-monomer percentages were assessed using a box-Behnken design. Rheometry, SEM, FTIR and swelling was used to measure changes in material properties in simulated physiological conditions. Rheometry revealed gelation temperature of hydrogel materials could be modified with dimethyl-acrylamide co-monomer; however, final maximum mechanical properties remained unaffected. Increasing the weight % and clay % increased resultant mechanical properties from ∼500–2500 G' (Pa), increased viscosity, but retained the ability to flow through a 26G needle at 39°C.Introduction
Methods & Results
Mesenchymal stem cells (MSCs) are undergoing evaluation as a potential new therapy for immune and inflammatory-mediated conditions such as IVD degeneration (IDD). Both adipose (ASCs) and bone-marrow (BMSCs) derived MSCs have been widely used in this regard. The optimal tissue source and expansion conditions required to exploit the regenerative capacity of these cells are not yet fully elucidated. In addition the phenotypic response of transplanted cells to the disease environment is not well understood. In this study, ASCs and BMSCs were exposed to a combination of hypoxic conditioning and selected inflammatory mediators, conditions that mimic the microenvironment of the degenerate IVD, in an effort to understand their therapeutic potency for Donor-matched ASCs and MSCs were pre-conditioned with either IL-1β (10ng/ml) or TNFα (10ng/ml) for 48 hours under hypoxic conditions (5% O2). Conditioned media was collected and 45 different immunomodulatory proteins were analysed using human magnetic Luminex® assay. Secreted levels of several key cytokines and chemokines, both pro- and anti-inflammatory, were significantly upregulated in ASCs and BMSCs following the conditioning regime. Under all conditions tested, ASCs expressed significantly higher levels of IL-4, IL-6, IL-10, IL-12, TGF-α, and GCSF compared to BMSCs. Pre-conditioning with TNFα resulted in significantly higher levels of IL-10 while preconditioning with IL-1β resulted in higher levels of IL-6, IL-12 and GCSF.Background
Methods and Results
Our study establishes a rabbit model of disc degeneration which requires neither a chemical nor physical injury to the disc. Disc degeneration similar to that seen in man was created at levels proximal (L4-L5) and caudal (L7-S1) to a simulated lumbar fusion and was studied for up to nine months after arthrodesis. Loss of the normal parallel arrangement of collagen bundles within the annular lamellae was observed in intervertebral discs adjacent to the fusion at three months. By six months there was further disorganisation as well as loss of distinction between the lamellae themselves. By nine months the structure of the disc had been replaced by disorganised fibrous tissue, and annular tears were seen. There was an initial cellular proliferative response followed by loss of chondrocytes and notochordal cells in the nucleus pulposus. Degeneration was accompanied by a decrease in the monomer size of proteoglycans. Narrowing of the disc space, endplate sclerosis and the formation of osteophytes at adjacent disc spaces were observed radiologically.
Intervertebral discs (IVDs) are fibrocartilagenous ovoids located between the vertebral bodies of the spine that provide the sole source of flexibility in that structure. IVDs are clinically very important as degeneration has been shown to be strongly associated with lower back pain, sciatica, and disc herniation: potentially disabling conditions that affect a very large section of the UK population. The aetiology of disc degeneration is poorly understood although upregulation of matrix metalloproteinase (MMP) activity is thought to be involved. Degradation products of the extra-cellular matrix are known to increase MMP production and activity in other tissues. This project concentrated on examining the effects of degredation products of elastin. Elastin fragments (κ-elastin peptides) have been shown to upregulate mRNA levels and increase expression of pro-MMP-1 in human skin fibroblasts, cells that are thought to be similar to those residing in the annulus fibrosus of intervertebral discs. This study examined their effect on disc cells and on skin fibroblasts. Total MMP-2 and -7 activity produced by cells extracted from the annulus fibrosus of bovine intervertebral disc cells and cultured for 24 hours with 0–300μg/ml κ-elastin was determined using fluorimetric and zymographic analyses. κ-elastin was prepared from bovine ligamentum nuchae or bovine intervertebral discs. Culture with κ-elastin prepared from bovine ligamentum nuchae caused skin and disc cell potential pro-MMP-2 activity to increase in a dose-dependent manner; the potential pro-MMP-2 activity of both cell types is more than doubled when cultured with 300μg/ml κ-elastin. These findings suggest that in the bovine disc, matrix breakdown may cause a feedback loop with degraded elastin stimulating disc cells to increase production of pro-MMP-2, with the possibility of further degrading elastin and other proteins and contributing to IVD breakdown.
We studied 52 patients, each with a lumbosacral transitional vertebra. Using MRI we found that the lumbar discs immediately above the transitional vertebra were significantly more degenerative and those between the transitional vertebrae and the sacrum were significantly less degenerative compared with discs at other levels. We also performed an anatomical study using 70 cadavers. We found that the iliolumbar ligament at the level immediately above the transitional vertebra was thinner and weaker than it was in cadavers without a lumbosacral transitional vertebra. Instability of the vertebral segment above the transitional vertebra because of a weak iliolumbar ligament could lead to subsequent disc degeneration which may occur earlier than at other disc levels. Some stability between the transitional vertebra and the sacrum could be preserved by the formation of either an articulation or by bony union between the vertebra and the sacrum through its transverse process. This may protect the disc from further degeneration in the long term.
T2D is postulated to be an important aetiological factor for lumbar disc degeneration (LDD), which itself has a well documented relationship with low back pain. Obesity increases risk of both T2D, low back pain and LDD. Connective tissue modification has been reported in hyperglycaemia, but the epidemiology of LDD in T2D has not been described to date. A population sample of unselected same-sex adult twin pairs was studied who had attended a spine MR study and completed general health questionnaires defining T2D by self-report. LDD had been coded as the sum of five lumbar discs coded (0–3) for each of height, signal intensity, disc bulge and anterior osteophytes. Risk factors for LDD included age, body-mass index (BMI), sex, alcohol consumption and smoking. Mean age of the 1011 participants = 54 years (sd=8), mean BMI=25 kg/m2 (sd=4), 95% female. The prevalence of T2D was 6%. Twins with T2D had increased BMI (27 vs 25 kg/m2, p<0.001) and were older (59 vs 54 years, p<0.001). LDD score in T2D was significantly higher in diabetic cases than controls (14.9 vs 13.1, p=0.04) in univariable analysis. In multivariable analysis inclusion of age and BMI abrogated the effect of T2D.Background and aims:
Methods and results:
Current strategies to treat back pain address the symptoms but not the underlying cause. Here we are investigating a novel hydrogel material (NPgel) which can promote MSC differentiation to Nucleus pulposus cells. Current hMSCs were encapsulated in NPgel and cultured for 4 weeks under hypoxia (5%) with ± calcium (2.5mM and 5.0mM CaCl2), IL-1β and TNFα either individually or in combination to mimic the degenerate microenvironment. Cell viability was assessed by Alamar blue assay. Histological and immunohistochemical analysis investigated altered matrix and matrix degrading enzyme expression.Introduction
Methods
Degenerate disc disease is a major cause of low back pain, yet its aetiology is still poorly understood. The intervertebral disc is the largest avascular structure in the body. Cells of the nucleus pulposus, therefore, rely on diffusion of oxygen &
nutrients down concentration gradients from peripheral vessels in the cartilage end-plates. Thus, there is a low oxygen tension and cellular respiration is largely anaerobic. The purpose of this study was to examine the effects of inflammation, hypoxia and acidosis on degeneration and pro-inflammatory mediator production in virgin porcine nucleus pulposus cultures. Intervertebral discs were harvested from normal 6-month old agricultural pigs slaughtered for other purposes. Nucleus pulposus was contained within the annulus until further dissection under sterile conditions in the laboratory was performed. Nucleus pulposus was harvested, diced and divided into 200mg samples. Samples were incubated under optimal conditions. Discs were cultured in 5μg/ml E. coli lipopolysaccharide, in a hypoxic environment or at low pH. IL-6, IL-8 and LDH assays were performed by ELISA, in accordance with manufacturer’s instructions. Time and dose-response curves were generated for each experiment (results not shown). Results at 72 hours incubation are tabulated below: These results confirm that nucleus pulposus is a biochemically active tissue capable of producing pro-inflammatory mediators in response to environmental stresses. IL-6 and IL-8 are both involved in the inflammatory cascade, causing chemotaxis of neutrophils and macrophages to the area. IL-8 itself causes hyperalgesia. Acidotic and inflammatory conditions, but not hypoxia, stimulated cytokine release. This may indicate a protective reduction in cellular activity in reduced oxygen environments. Necrosis, as measured by LDH production, was negligible.
Anatomisches Institut der Georg-August-Universität Göttingen, Germany Biomedical Research Centre, Dept. of Orthopaedic Surgery, Academic Hospital, Pretoria, South Africa To date, no animal model for disc degeneration has gained much acceptance, mostly due to the fact that most animals are quadrupeds and thus lack basic biomechanical characteristics of human spines. An adequate model would be of invaluable interest for degeneration related research. In a standardized series of animal experiments in 18 adult Minipigs and 20 adult Cercopithecus aethiops monkeys all animals obtained nucleotomy in one lumbar FSU from a ret-roperitoneal approach and were sacrificed at last 24 weeks afterwards. The Minipigs were x-rayed at time of sacrifice, the monkeys prior to operation and at termination of the experiment. Vice versa, the Minipigs obtained intradiscal pressure recordings at these occasions. The Minipig spines were formol fixed whereas the monkey spines were harvested after perfusion with PBS, fresh frozen, and obtained CT and MRI scans prior to thawing, fixation and comprehensive histological evaluation. The lumbar FSU of Minipig and Cercopithecus mainly consists of the same elements as in man. There are certain differences concerning the porcine endplates which ossify as an epiphyseal-like formation with ossification starting in its center, different from the so-called “Randleiste”. Whereas the operative procedure in the Minipigs came in handy, in the Cercopithecus monkey it proved to be demanding, though feasible, due to relatively wide transverse processes and thick psoas muscle structures. The psoas could not be easily detached and needed to be split instead, thus directly exposing the segment nerves. The histological, standard radiological, CT, MRI, and mechanical observations were very similar to those which can be made during the natural aging process of the disc in man. Both animal models are recommendable for further research: Cercopithecus FSUs are more difficult to expose. Logistic reasons may favour Minipigs in Europe. In case of fusion related experiments the use of primates yet seems inevitable.
We attempted to correlate the findings of MRI and discography in patients with low back pain, examining 108 lumbar intervertebral discs in 33 consecutive patients. MRI results were assessed from the intensity and shape of the signal obtained from the central part of the disc. Discography was classified according to the pattern of contrast material, the pressure accepted and the pain reproduced. All discs which were abnormal on MRI had altered patterns on discography, but 18 of the 60 discs with normal MRI had abnormal discograms. Of 39 asymptomatic discs, 33 had normal MRI signals and 24 had normal discograms. None of the 15 discs showing severe degeneration on MRI sustained high levels of intradiscal pressure, but only six of the 60 discs giving normal MRI had low pressure. With current techniques, discography is more accurate than MRI for the detection of annular pathology: a normal MRI does not exclude significant changes in the peripheral structure of the intervertebral disc which can produce low back pain.
For phase angle (approximate magnitude 5°), no significant overall effects due to degeneration were found across any loading direction (P>
0.2). ANOVA analyses on maximum/minimum principal strains found no significant effect due to disc grade (P>
0.063). However, a small number of significant effects due to disc grade were found at particular strain gauge locations for the isolated disc in flexion, the intact FSU in extension, and the intact FSU/isolated disc in right lateral bending.
The non-significant small phase angles suggest that the disc behaves more like an elastic solid than a poroelastic material, and that dehydration associated with degeneration does not adversely affect damping. Principal strains were not significantly affected by disc degeneration overall, suggesting that the cortical shell adjacent to the disc-endplate boundary maintains a relatively homeostatic condition, with more dramatic architectural changes probably occurring within the trabecular bone. Applications of this research include providing important validation data for analytical/finite element models of the intact FSU and isolated disc segment, and a better understanding of the magnitudes of cortical strains that need to be maintained in order to avoid damaging vertebral bone stress-shielding effects after treatments for disc degeneration.
Disc degeneration is often scored using macroscopic and microscopic scoring systems. Although reproducible, these scores may not accurately reflect declining function in a degenerated disc. Accordingly, we compared macroscopic and microscopic degeneration scores with measurements of disc function. Thirteen cadaveric motion segments (62–93 yrs) were compressed to 1kN while a pressure-transducer was pulled across the mid-sagittal diameter of the disc. Resulting stress profiles indicated intradiscal pressure (IDP), and maximum stress in the anterior (MaxStress_Ant) and posterior (MaxStress_Post) annulus. Macroscopic Introduction
Methods
We investigated the relationship between spinopelvic
parameters and disc degeneration in young adult patients with spondylolytic
spondylolisthesis. A total of 229 men with a mean age of 21 years
(18 to 26) with spondylolytic spondylolisthesis were identified.
All radiological measurements, including pelvic incidence, sacral
slope, pelvic tilt, lumbar lordosis, sacral inclination, lumbosacral
angle (LSA), and sacrofemoral distance, were calculated from standing
lateral lumbosacral radiographs. The degree of intervertebral disc
degeneration was classified using a modified Pfirrmann scale. We
analysed the spinopelvic parameters according to disc level, degree
of slip and disc degeneration. There were significant positive correlations between the degree
of slip and pelvic incidence (p = 0.009), sacral slope (p = 0.003)
and lumbar lordosis (p = 0.010). The degree of slip and the LSA
were correlated with disc degeneration (p <
0.001 and p = 0.003,
respectively). There was also a significant difference between the
degree of slip (p <
0.001) and LSA (p = 0.006) according to the
segmental level of disc degeneration. Cite this article:
The presence of facet tropism has been correlated with an elevated susceptibility to lumbar disc pathology. Our objective was to evaluate the impact of facet tropism on chronic lumbosacral discogenic pain through the analysis of clinical data and finite element modelling (FEM). Retrospective analysis was conducted on clinical data, with a specific focus on the spinal units displaying facet tropism, utilizing FEM analysis for motion simulation. We studied 318 intervertebral levels in 156 patients who had undergone provocation discography. Significant predictors of clinical findings were identified by univariate and multivariate analyses. Loading conditions were applied in FEM simulations to mimic biomechanical effects on intervertebral discs, focusing on maximal displacement and intradiscal pressures, gauged through alterations in disc morphology and physical stress.Aims
Methods
Cite this article: