Cartilaginous Endplate Research Articles

Exogenous Stimulation of Human Intervertebral Disc Cells in 3-Dimensional Alginate Bead Culture With BMP2 and L51P: Cytocompatibility and Effects on Cell Phenotype.

ObjectiveSpinal fusion surgery is a common treatment modality for various pathologic conditions of the spine. The bone morphogenetic protein 2 (BMP2) analogue L51P acts as a general inhibitor of BMP antagonists, whereas it shows a weak affinity for BMP type I receptor. It is suggested that L51P applied in bone disorders might prevent side effects of highly concentrated BMP dosage applications in the order of milligrams. The objective of this study was to investigate the effects of L51P and BMP2 on intervertebral disc cells (IVDCs), i.e. on nucleus pulposus cells, on annulus fibrosus cells (AFCs), and on cartilaginous endplate cells (CEPCs), respectively, in 3-dimensional (3D) culture. MethodsLow-passage primary IVDCs were cultured in 3D alginate bead culture and exposed to 100-ng/mL BMP2 and/or L51P for 21 days. Here, we analyzed glycosaminoglycan (GAG) and DNA content and further performed gene expression analysis for major matrix genes. ResultsAFCs and cartilaginous CEPCs stimulated with each 100-ng/mL L51P and BMP2, showed a significant upregulation in GAG (AFCs: p = 0.00347 and CEPCs: p = 0.0115) and DNA production (AFCs: p = 0.0182 and CEPCs: p = 0.0179) compared to control. ConclusionThese results allow first insights into the behavior of IVDCs upon L51P stimulation.

Intervertebral disc and endplate cell characterisation highlights annulus fibrosus cells as the most promising for tissue-specific disc degeneration therapy.

Degenerative processes of the intervertebral disc (IVD) and cartilaginous endplate lead to chronic spine pathologies. Several studies speculated on the intrinsic regenerative capacity of degenerated IVD related to the presence of local mesenchymal progenitors. However, a complete characterisation of the resident IVD cell populations, particularly that isolated from the endplate, is lacking. The purpose of the present study was to characterise the gene expression profiles of human nucleus pulposus (NPCs), annulus fibrosus (AFCs) and endplate (EPCs) cells, setting the basis for future studies aimed at identifying the most promising cells for regenerative purposes. Cells isolated from NP, AF and EP were analysed after in vitro expansion for their stemness ability, immunophenotype and gene profiles by large-scale microarray analysis. The three cell populations shared a similar clonogenic, adipogenic and osteogenic potential, as well as an immunophenotype with a pattern resembling that of mesenchymal stem cells. NPCs maintained the greatest chondrogenic potential and shared with EPCs the loss of proliferation capability during expansion. The largest number of selectively highly expressed stemness, chondrogenic/tissue-specific and surface genes was found in AFCs, thus representing the most promising source of tissue-specific expanded cells for the treatment of IVD degeneration.

Diagnostic value and clinical significance of magnetic resonance imaging with the FS-PD-TSE sequence in diagnosing lumbar cartilaginous endplate failure.

The aims were to use magnetic resonance imaging (MRI) to compare the efficacy of fat-suppressed proton-density turbo-spin-echo (FS-PD-TSE) images and T1-weighted (T1WIs) and T2-weighted images (T2WIs) in identifying cartilaginous endplate failure (CEF), and to propose a modified Rajasekaran classification based on the FS-PD-TSE sequence. Thirty-one lumbar disc herniation (LDH) cases were enrolled. Totally, 155 discs and 310 endplates were evaluated by MRI with T1W, T2W, and FS-PD-TSE sequences. Disc degeneration (DD), LDH grades, and the total endplate score (TEPS) of CEF were evaluated. Chi-square, Spearman rank correlation analysis, and multiclass logistic regression were used to compared the sensitivity in diagnosing CEF. A modified Rajasekaran classification based on FS-PD-TSE sequence was established to diagnose CEF. The multiclass logistic regression model was used to analyse the relationship between modified Rajasekaran classification and DD and LDH. There were 117 (75.5%) segments with CEF in T1WIs, 115 (74.2%) in T2WIs, and 127 (81.9%) in FS-PD-TSE, respectively. Chi-square test showed FS-PD-TSE images were more sensitive than T1WIs and T2WIs (P < 0.05). Spearman rank correlation analysis revealed a significant correlation between TEPS and LDH and DD in T1WIs, T2WIs, and FS-PD-TSE images (P < 0.05). A multiclass logistic regression model showed that the incidence of DD and LDH significantly increased accordingly with increases in modified Rajasekaran classification (P < 0.05). The FS-PD-TSE sequence has high diagnostic value for lumbar CEF. CEF is a risk factor for LDH. The new classification for lumbar CEF based on the FS-PD-TSE sequence has good predictive ability for LDH and DD. These slides can be retrieved under Electronic Supplementary Material.

Small molecule inhibitor of TAK1 ameliorates rat cartilaginous endplate degeneration induced by oxidative stress in vitro and in vivo

Small molecule inhibitor of TAK1 ameliorates rat cartilaginous endplate degeneration induced by oxidative stress in vitro and in vivo

The Immune Privilege of the Intervertebral Disc: Implications for Intervertebral Disc Degeneration Treatment.

The intervertebral disc (IVD) is the largest avascular organ of the body. It is composed of three parts: the nucleus pulposus (NP), the annulus fibrosus (AF) and the cartilaginous endplate (CEP). The central NP is surrounded by the AF and sandwiched by the two CEPs ever since its formation. This unique structure isolates the NP from the immune system of the host. Additionally, molecular factors expressed in IVD have been shown inhibitive effect on immune cells and cytokines infiltration. Therefore, the IVD has been identified as an immune privilege organ. The steady state of immune privilege is fundamental to the homeostasis of the IVD. The AF and the CEP, along with the immunosuppressive molecular factors are defined as the blood-NP barrier (BNB), which establishes a strong barrier to isolate the NP from the host immune system. When the BNB is damaged, the auto-immune response of the NP occurs with various downstream cascade reactions. This effect plays an important role in the whole process of IVD degeneration and related complications, such as herniation, sciatica and spontaneous herniated NP regression. Taken together, an enhanced understanding of the immune privilege of the IVD could provide new targets for the treatment of symptomatic IVD disease. However, the underlying mechanism above is still not fully clarified. Accordingly, the current study will extensively review and discuss studies regarding the immune privilege of the IVD.

A review of discogenic pain management by interventional techniques.

This review article describes the various image guided interventional techniques used for treating chronic backache attributed to disc related pathologies. With the aim of minimum invasion and maximum relief, these procedures comprise predominantly of annuloplasty and disc decompression via different mechanisms. Newer therapies are discussed in this review article with the objective of restoring disc height and its biomechanical function by substitution of biochemical constituents, regeneration of cartilaginous end plate and finally artificial disc implantation.

Parkin and Nrf2 prevent oxidative stress-induced apoptosis in intervertebral endplate chondrocytes via inducing mitophagy and anti-oxidant defenses

AimsEndplate chondrocyte apoptosis is an important contributor to the pathogenesis of cartilaginous endplate (CEP) degeneration that leads to the initiation and development of intervertebral disc degeneration (IDD). In this study, we hypothesized that Parkin-mediated mitophagy and nuclear factor E2-related factor 2 (Nrf2)-mediated antioxidant system played an important role in endplate chondrocyte survival under pathological conditions. Materials and methodsHuman endplate chondrocytes were stimulated with H2O2 to mimic pathological conditions. Western blotting, immunofluorescence staining, and flow cytometry were applied to detect the indicators related to mitochondrial dynamics, mitophagy, Nrf2 signaling, and apoptosis. The puncture-induced rat models were established to evaluate the changes in vivo. Key findingsOur results showed that H2O2 induced oxidative stress, mitochondrial dysfunction, and apoptosis in endplate chondrocytes. These H2O2-induced detrimental effects were inhibited by pretreatment with the mitochondria-targeted antioxidant Mito-TEMPO. In addition, mitochondrial dynamics, Parkin-mediated elimination of dysfunctional mitochondria, and Nrf2-mediated antioxidant system were promoted by H2O2. Knockdown of Parkin or Nrf2 increased H2O2-induced detrimental effects. Moreover, upregulation of Parkin and Nrf2 by polydatin protected endplate chondrocytes against H2O2-induced mitochondrial dysfunction, oxidative stress, and apoptosis. Finally, puncture-induced rat models showed that polydatin exerted a protective effect on CEP and disc degeneration. SignificanceTargeting Parkin and Nrf2 to improve mitochondrial homeostasis, redox balance and endplate chondrocyte survival may represent a potential therapeutic strategy for preventing IDD.

Spatial distribution of type II collagen gene expression in the mouse intervertebral disc.

Genetic tools such as the Cre‐Lox reporter system are powerful aids for tissue‐specific cell tracking. For example, it would be useful in examining intervertebral disc (IVD) cell populations in normal and diseased states. A Cre recombinase and its recognition site, loxP have been adapted from the bacteriophage for use in genetic manipulation. The reporter mice used here express the red fluorescent protein, tdTomato with flanking LoxP sites (Rosa26 TdTomato mice). We compared two different Collagen type II (Col2) promoter constructs that drive Cre‐recombinase expression in mice: (a) Col2‐Cre, which allows constitutive Cre‐recombinase expression under the control of the Col2 promoter/enhancer and (b) Col2‐CreER, which contains a shorter promoter/enhancer region than Col2‐Cre, but has human estrogen binding elements that bind tamoxifen, resulting in Cre‐recombinase expression. The goal of the study is to characterize Cre‐recombinase distribution pattern in Col2‐Cre and Col2‐CreER mice using tdTomato as reporter in the spine. The expression patterns of these two mice were further compared with Col2 gene expression in the native mouse NP and AF tissues by real‐time PCR. We crossed Col2‐Cre mice or Col2‐CreER mice with the tdTomato reporter mice, and compared the tdTomato expression patterns. Col2‐CreER/tdTomato mice were injected with tamoxifen at postnatal day 7 to activate the Cre‐recombinase. TdTomato in the constitutively active Col2‐Cre mice was detected in the nucleus pulposus (NP), the entire annulus fibrosus (AF), and in cartilaginous endplate and growth plate cells in the lower lumbar and coccygeal spine. In contrast, when Col2‐CreER activity was induced by tamoxifen at P7, tdTomato was limited to the inner AF, and was absent from the NP. We have described the differences in Col2 reporter gene expression, in Col2‐Cre/tdTomato and Col2‐Cre‐ER/tdTomato mouse IVD. The information provided here will help to guide future investigations of IVD biology.

Oswestry Disability Index scores correlate with MRI measurements in degenerating intervertebral discs and endplates.

Low back pain (LBP) is a widespread problem and the leading cause of disability worldwide. While the cause of LBP is multifactorial, several studies suggested that inflammatory mediators in damaged subchondral plates of degenerating discs may lead to chemical sensitization and mechanical stimulation, eventually causing pain. The goal of this study was to explore associations between such changes and LBP-related disability using dynamic contrast-enhanced MRI. Thirty-two patients diagnosed with nonspecific LBP and 24 healthy control subjects were studied with dynamic contrast-enhanced (DCEMRI) MRI and T1r (spin-lattice relaxation in the rotating frame) acquisitions. DCEMRI enhancement in disc endplate regions and average T1ρ measurements in the nucleus pulposus were extracted. The LBP patients were grouped based on their Oswestry Disability Index (ODI) scores and associations between MRI measurements and ODI scores were analyzed. Significant associations were found between ODI scores and DCEMRI enhancement in the cartilaginous endplate regions of the most degenerated discs. ODI scores also correlated with T1ρ measurements in the nucleus pulposus of degenerating discs. DCEMRI enhancement in the cartilaginous endplate regions and lower T1ρ measurements in the nucleus pulposus (NP) were associated with greater disability that is related to low back pain as reported on the ODI. This complements earlier reports suggesting a link between LBP and endplate degeneration. Further studies are needed to validate these findings. Our findings indicated that dynamic contrast-enhanced MRI signal enhancement in the cartilaginous endplate regions were associated with greater disability related to low back pain. This signal enhancement might be an indication of inflammatory changes in disc endplate regions. Therefore, advanced quantitative imaging techniques like the ones presented in this study might be needed to complement conventional radiological evaluations to identify the subset of patients who could potentially benefit from novel therapies directed towards treating the disc endplate regions.

Imaging diagnosis for intervertebral disc.

Various functional magnetic resonance imaging (MRI) techniques have been investigated in recent years and are being used in clinical practice for the patients with low back pain (LBP). MRI is an important modality for diagnosing intervertebral disc (IVD) degeneration. In recent years, there have been several reported attempts to use MRI T2 mapping and MRI T1ρ mapping to quantify lumbar disc degeneration. MRI T2 mapping involves digitizing water content, proteoglycan content, and collagen sequence breakdown as relaxation times (T2 values) at each site. These digitized values are used to create a map, that is, then used to quantitatively evaluate the metabolite concentrations within IVD tissues. MRI T2 mapping utilizes the T2 relaxation time to quantify moisture content and the collagen sequence breakdown. MRI T1ρ mapping digitizes water molecule dispersion within the cartilaginous matrix to evaluate the degree of cartilaginous degeneration. Magnetic resonance spectroscopy is a less‐invasive diagnostic test that provides biochemical information. Adequate analysis of the IVD has not yet been performed, although there are indications of a relationship between the adipose content of the multifidus muscle in the low back and LBP. The ultra short TE technique has been recently used to investigate lumbar cartilaginous endplates. Unlike diagnosis based on contrast‐enhanced images of the IVD, which depends on the recurrence of pain that is determined subjectively, MRI‐based diagnosis is less‐invasive and based on objective imaging findings. It is therefore expected to play a key role in the diagnostic imaging of IVD conditions in the future.

Intervertebral disc swelling maintains strain homeostasis throughout the annulus fibrosus: A finite element analysis of healthy and degenerated discs

Tissues in the intervertebral disc have a large capacity to absorb water, partially due to the high glycosaminoglycan (GAG) content, which decreases linearly from the nucleus pulposus (NP) in the center to the outer annulus. Our recent work showed that fiber network and GAG distribution contributes to development of residual stresses and strains that were compressive in the inner annulus to tensile in the outer annulus. GAG loss in the inner annulus, as observed with early to moderate degeneration, reduced swelling capacity and circumferential-direction stress by over 50%. However, our previous model was not capable of evaluating interactions between the NP and annulus fibrosus (AF) during swelling. In this study, we evaluated the effect of degeneration (GAG content or swelling capacity) on residual stress development throughout the disc. Simulations of moderate to severe degeneration showed a 40% decrease in NP swelling capacity, with a 25% decrease in AF and cartilaginous endplate swelling. Together, these changes in tissue swelling resulted in a decrease in NP pressure (healthy = 0.21 MPa; severe degeneration = 0.03 MPa) that was comparable to observations in human discs. There was a 60% decrease in circumferential-direction residual deformations with early degeneration. Radial-direction stretch switched from compressive to tensile with degeneration, which may increase the risk for tears or delamination. Degeneration had a significant impact on residual stress/stretch and fiber stretch in the posterior AF, which is important for understanding herniation risk. In conclusion, degenerative changes in disc geometry and intradiscal deformations was recreated by only altering NP and AF GAG composition. Since most computational models simulate degeneration by altering material stiffness, this work highlights the importance of directly simulating biochemical composition and distribution to study disc biomechanics with degeneration. Statement of significanceTissues in the intervertebral disc have a large swelling capacity, due to its high glycosaminoglycan content. Our recent work demonstrated the importance of fiber network and glycosaminoglycan distribution residual stresses and strains development. In this study, we evaluated the effect of swelling on intradiscal deformations between the nucleus pulposus and annulus fibrosus. We also investigated the effect of degenerative glycosaminoglycan loss on swelling-based intradiscal deformations of the intact disc and its subcomponents. Decreases in nucleus glycosaminoglycan content resulted in morphological changes observed with degenerated discs and may help to explain mechanisms behind the increases in annular tears and mechanical dysfunction with degeneration.

Vertebrae, Vertebral End Plates, and Disks: Concepts and Specific Pathologies.

Vertebral end plates cover the osseous vertebral body. The integrity of the cartilaginous end plates is of great importance for the entire vertebral segment because the vascularized end plate provides the nutrition for the avascular disk. Yet several pathologies may occur at these end plates at the embryonic stage, in childhood to adolescence (e.g., ossification and segmentation disorders of the spine, persistent notochord, slippage of the growth plate), as well as in the mature spine of an adult (degenerative disk disease), that may impact the integrity of the cartilaginous end plate and therefore lead to severe diseases of the spine. This article reviews specific congenital, developmental, and degenerative disorders of the vertebral end plate as well as both established and newly introduced imaging techniques, such as ultrashort echo time imaging based on magnetic resonance imaging, that are suitable for imaging of the end plate.

T140 Inhibits Apoptosis and Promotes Proliferation and Matrix Formation Through the SDF-1/CXC Receptor-4 Signaling Pathway in Endplate Chondrocytes of the Rat Intervertebral Discs

Cartilaginous endplate (CEP), a thin layer of hyaline cartilage located between the vertebral endplate and nucleus pulposus, transports the nutrient into the disc. The objective of this study was to evaluate the influence of T140 (polyphemusin II-derived peptide) on the CEP cell growth, apoptosis, and the matrix formation via the stromal cell-derived factor-1 (SDF-1)/cysteine X cysteine (CXC) receptor-4 (CXCR4) signaling pathway. Sprague-Dawley rats were euthanized by cervical dislocation and dissected for the isolation and the appraisal of CEP cells that were extracted from the endplate in rat intervertebral discs and were then added with different concentrations of reagents (SDF-1 and T140). The effect of T140 on CEP cell proliferation and apoptosis were analyzed. The messenger RNA (mRNA) and protein expressions of CXCR4, prominin-1, proteoglycans, type II collagen, B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein were analyzed by reverse transcription quantitative polymerase chain reaction and Western blot analysis. T140 promoted the proliferation of CEP cells and inhibited the apoptosis of CEP cells. Additionally, T140 suppressed the mRNA and protein expression of CXCR4, prominin-1, and Bcl-2 associated X protein, and increased the mRNA and protein expression of proteoglycans, type II collagen, and Bcl-2. T140 promotes the proliferation and matrix formation and inhibits the apoptosis of CEP cells by blocking the SDF-1/CXCR4 signaling pathway invitro, which provides a certain therapeutic effect on the degeneration of intervertebral discs.

Inhibition of EZH2 Ameliorates Cartilage Endplate Degeneration and Attenuates the Progression of Intervertebral Disc Degeneration via Demethylation of Sox-9

Backgroud: Cartilaginous endplate (CEP) degeneration is considered as one of the major causes of intervertebral disc degeneration (IVDD) which causes low back pain. Recent studies have proved that epigenetic alteration is involved in a variety of diseases. This work explored the role of histone methyltransferase enhancer of zeste homologue 2 (EZH2) in CEP degeneration, as well as its underlying epigenetic mechanisms, and confirmed the effect of EZH2 knockdown on delaying IVDD development. Methods: Western blotting, immunofluorescence staining, and ChIP assay were applied to demonstrate the molecular mechanism of EZH2 in CEP tissue. The therapeutic potential of EZH2 was investigated using puncture-induced rat models. Findings: The EZH2 expression was upregulated in human and rat CEP tissue. It was also found that the overexpression of EZH2 suppressed the expression of Collagen II, aggrecan and Sox-9, and promoted the expression of ADTAMTS5 and MMP13 in rat endplate chondrocytes (EPCs), which could be reversed by EZH2 silencing. The correlation between EZH2 and Sox-9 was further explored, while overexpression of Sox-9 could reverse the effect of EZH2 in rat EPCs. Moreover, inhibition of EZH2 upregulated the level of Sox-9 by demethylating H3K27me3 at Sox-9 promoter sites, revealing the regulatory mechanism of EZH2 on Sox-9. Meanwhile, puncture-induced rat models showed that EZH2 knockdown exerted a protective effect on CEP and disc degeneration. Interpretation: This study reveals that EZH2 inhibition is a promising strategy for mitigating the symptoms and progression of IVDD. Funding: This study was funded by the Natural Science Foundation of Zhejiang Province (Y16H060034). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: All surgical interventions, treatments and postoperative animal care procedures were strictly performed in accordance with the guidelines for Animal Care and Use outlined by the Committee of Wenzhou Medical University. Human CEP tissue collection and experiments that involved human CEP were approved by the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University Ethics Committee and followed the guidelines of the Helsinki Declaration.

Intervertebral Disc Swelling Maintains Strain Homeostasis Throughout the Annulus Fibrosus: A Finite Element Analysis of Healthy and Degenerated Discs

Tissues in the intervertebral disc have an excellent capacity to absorb water, largely due to the high glycosaminoglycan (GAG) content, which decreases linearly from the nucleus pulposus (NP) in the center to the outer annulus. Our recent work showed that fiber network and GAG distribution contributes to development of residual stresses and strains that were compressive in the inner annulus to tensile in the outer annulus. GAG loss in the inner annulus, as observed with early to moderate degeneration, reduced swelling capacity and circumferential-direction stress by over 50%. However, our previous model was not capable of evaluating interactions between the NP and annulus fibrosus (AF) during swelling. In this study, we evaluated the effect of degeneration (GAG content or swelling capacity) on residual stress development throughout the disc. Simulations of moderate to severe degeneration showed a 40% decrease in NP swelling capacity, with a 25% decrease in AF and cartilaginous endplate swelling. Together, these changes in tissue swelling resulted in a decrease in NP pressure (healthy = 0.21MPa; severe degeneration = 0.03MPa) that was comparable to observations in human discs. There was a 60% decrease in circumferential-direction residual deformations with early degeneration. Radial-direction stretch switched from compressive to tensile with degeneration, which may increase the risk for tears or delamination. Degeneration had a significant impact on residual stress/stretch and fiber stretch in the posterior AF, which is important for understanding herniation risk. In conclusion, relatively large changes in local strains occurred with degeneration, which may contribute to further degenerative remodeling.

Genetic Predisposition to Symptomatic Lumbar Disk Herniation in Pediatric and Young Adult Patients.

Case-control whole-genome sequencing analysis of a highly select, young cohort with symptomatic lumbar disk herniation (LDH) compared with the standard variation in a large reference population. To assess genetic influences predisposing pediatric and young adult patients to symptomatic LDH. LDH has traditionally been attributed to natural weakening or mechanical insult, but recent literature supports a potential genetic influence. Young patients with symptomatic, clinically confirmed LDH who underwent surgical treatment were included. Patients were younger than the average age of presentation, limiting the influence of environmental risks. DNA collected from these patients was compared with a reference genome (1000 Genomes Project). A genome-wide association study using whole-genome sequencing was used to characterize genetic mutations potentially associated with LDH. Among the 61 candidate genes flagged, 20 had missense mutations in 2 or more LDH cases. Missense mutations in collagen-encoding genes were observed in 12 of 15 patients (80%). A potential association with clinical presentation was indicated by odds ratios of key single-nucleotide polymorphism (SNP) variants in genes that encode collagen. Relative to the reference population, the LDH cohort demonstrated two statistically significant SNP variants in the gene encoding for aggrecan, a protein that facilitates load-bearing properties in the cartilaginous end plate. Aggrecan genes SNPs rs3817428 and rs11638262 were significantly associated with decreased odds of symptomatic LDH: odds ratio 0.05 (0.02-0.11) and 0.04 (0-0.26), respectively (P < 1 × 10 for both). These results suggest that collagen-encoding variants may be a genetic risk factor for LDH. They also shed new light on the role of variants that impact aggrecan, which sustains the cartilaginous end plate. Genetic predisposition to LDH may therefore be related to a multimodal combination of mutations that affect the nucleus pulposus, annulus fibrosus, and the cartilaginous end plates. 4.

β‑catenin signalling inhibits cartilage endplate chondrocyte homeostasis invitro.

Cartilaginous endplate degeneration serves a key role in the process of intervertebral disc (IVD) degeneration, however, effective therapies are hindered by an incomplete understanding of the mechanisms that underlie cartilage endplate (CEP) homeostasis and degeneration. Wnt/β-catenin signalling has been reported as a major factor in regulating biological processes. Whether Wnt/β-catenin signalling engages in CEP homeostasis has not yet been investigated. The present study aimed to assess the function of CEP cells via the activation of Wnt/β-catenin signalling to examine and promote the mechanism of degeneration of CEP in vitro. Rat CEP cells were confirmed to exhibit a chondrocytic phenotype by toluidine blue staining. The increased number of senescence-associated β-galactosidase (SA-β-gal)-positive cells and reduced cellular proliferation were investigated in the presence of a β-catenin inhibitor, and the inhibitor improved the trend of senescence. An increased number of apoptotic cells was detected by lithium chloride treatment, and inhibiting Wnt/β-catenin signalling protected the cells from apoptosis. Expression of the catabolic enzymes, metalloproteinase-13 and a disintegrin and metalloproteinase with thrombospondin motifs-5, and the decreased expression of aggrecan were also observed by Wnt/β-catenin signalling activation, and a Wnt/β-catenin signalling inhibitor decreased the expression of catabolic enzymes and increased the expression of aggrecan induced by Wnt/β-catenin signalling activation. Wnt/β-catenin signalling may provide potential strategies for preventing CEP degeneration.

Alpha 2-Macroglobulin as Dual Regulator for Both Anabolism and Catabolism in the Cartilaginous Endplate of Intervertebral Disc.

Basic science study. To illustrate supplemental alpha-2 macroglobulin (α2 M) has beneficial effects on cartilaginous endplates (CEPs) that may slow the progression of intervertebral disc (IVD) degeneration. CEPs play a vital role in progression of intervertebral disc degenerative diseases. However, the ideal and economic therapies for CEPs degeneration are still urgently required. Firstly, we confirmed degenerative CEP characters by H&E and Safranin O fast green staining and detected increasing level of α2 M and matrix metalloproteinase 13(MMP-13) in degenerative CEP by immunohistochemistry. Then, effects of exogenous α2 M on tumor necrosis factor alpha (TNF-α)-induced CEP catabolic enzyme and anabolic molecules were evaluated by qRT-PCR, Western blotting and ELISA in cultured CEP cells obtained from rats. Furthermore, suppression of α2 M on TNF-α-induced activation of NF-кB signaling pathway was measured by Western blotting and immunofluorescence. In addition, function of α2 M on TNF-α-treated ex vivo IVDs from rats lumbar IVDs was estimated by measuring the expression of MMP-13, Sox9, aggrecan, and type II collagen in CEP area. Compared with normal CEP, level of α2 M was slightly increased in CEP from degenerative patients, whereas MMP-13 was sharply elevated. In vitro, α2 M inhibited expression and activity of MMP-3 or MMP-13 in a dose-dependent manner in rat CEP cells stimulated by TNF-α. The α2 M refrained phosphorylation of IκBα and inhibited nuclear translocation of p65. Finally, supplemental α2 M reduced expression of MMP-13, and promoted expression of Sox9, aggrecan, and type II collagen in CEP area of ex vivo IVDs cultured with TNF-α. α2 M is not sufficiently produced to inactivate higher concentrations of catabolic factor MMP-13 found in the degenerated CEP. Supplemental α2 M protects against the progression of IVD degeneration by inhibiting effects of proinflammatory cytokines. N/A.

A histocytological and radiological overview of the natural history of intervertebral disk: from embryonic formation to age-related degeneration.

To elucidate the natural history of intervertebral disk (IVD) and characterize its embryonic beginnings and age-related degeneration. Coronal sections of embryonic (E13.5-neonatal) and postnatal (4-60-week-old) Sprague-Dawley rat IVD were stained by a series of histological stainings (hematoxylin and eosin, Alcian blue, Picrosirius red, Masson, Periodic acid-Schiff). Growth kinetics within embryonic IVD were evaluated by immunohistochemical staining of Ki67 and proliferating cell nuclear antigen. Postnatal maturation and degeneration of IVD were visualized on radiology by X-ray, CT, and MR imaging. During the formation of rat IVD, inner annulus fibrosus (AF) and cartilaginous endplate (CEP) shared similar cell density, extracellular matrix, and potential of growth kinetics; notochord provided increased and enlarged cytoplasmic vacuoles to generate nucleus pulposus (NP), part of which was retained within CEP. Postnatally, vacuolated notochord cells were reduced by devacuolation, while chondrocytic NP cells increased; cartilaginous layers of CEP were narrowed by vertebrae growth and secondary ossification; fibrotic portion of AF decreased as cartilaginous matrix accumulated and infiltrated outward. In aged and degenerated IVD, large longitudinal fissures were detected near the boundaries between inner and outer AF, whereas both reduced cellularity and accumulated cell clusters were evident within the dehydrated NP; only part of these histocytological changes could be reported on radiology. By showing that the natural history of IVD is orchestrated by a dynamic histocytological regulation, our study may facilitate better understanding of the developmental defects, cellular heterogeneity, age-related degenerative mechanisms, and biological regeneration of IVD. These slides can be retrieved under Electronic Supplementary Material.

Characterization of Cre recombinase mouse lines enabling cell type-specific targeting of postnatal intervertebral discs.

Cre/loxP technology is an important tool for studying cell type-specific gene functions. Cre recombinase mouse lines, including Agc1-CreERT2 , Col2a1-Cre; Col2a1-CreERT2 , Shh-Cre, Shh-CreERT2 , and Osx-Cre, have been proven to be valuable tools to elucidate the biology of long bones, yet the information for their activity in postnatal intervertebral disc (IVD) tissues was very limited. In this study, we used R26-mTmG fluorescent reporter to systematically analyze cell specificity and targeting efficiency of these six mouse lines in IVD tissues at postnatal growing and adult stages. We found that Agc1-CreERT2 is effective to direct recombination in all components of IVDs, including annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplate (CEP), upon tamoxifen induction at either 2 weeks or 2 months of ages. Moreover, Col2a1-Cre targets most of the cells in IVDs, except for some cells in the outer AF (OAF) and NP. In contrast, the activity of Col2a1-CreERT2 is mainly limited to the IAF of IVD tissues at either stage of tamoxifen injection. Similarly, Shh-Cre directs recombination specifically in all NP cells, whereas Shh-CreERT2 is active only in a few NP cells when tamoxifen is administered at either stage. Finally, Osx-Cre targets cells in the CEP, but not in the NP or AF of IVDs tissues at these two stages. Thus, our data demonstrated that all these Cre lines can direct recombination in IVD tissues at postnatal stages with different cell type specificity and/or targeting efficiency, and can, therefore, serve as valuable tools to dissect cell type-specific gene functions in IVD development and homeostasis.