Cartilaginous Endplate Degeneration Research Articles

Chondrocyte-targeted exosome-mediated delivery of Nrf2 alleviates cartilaginous endplate degeneration by modulating mitochondrial fission

BackgroundCartilaginous endplate (CEP) degeneration, which is an important contributor to intervertebral disc degeneration (IVDD), is characterized by chondrocyte death. Accumulating evidence has revealed that dynamin-related protein 1 (Drp1)-mediated mitochondrial fission and dysfunction lead to apoptosis during CEP degeneration and IVDD. Exosomes are promising agents for the treatment of many diseases, including osteoporosis, osteosarcoma, osteoarthritis and IVDD. Despite their major success in drug delivery, the full potential of exosomes remains untapped.Materials and methodsIn vitro and in vivo models of CEP degeneration were established by using lipopolysaccharide (LPS). We designed genetically engineered exosomes (CAP-Nrf2-Exos) expressing chondrocyte-affinity peptide (CAP) on the surface and carrying the antioxidant transcription factor nuclear factor E2-related factor 2 (Nrf2). The affinity between CAP-Nrf2-Exos and CEP was evaluated by in vitro internalization assays and in vivo imaging assays. qRT‒PCR, Western blotting and immunofluorescence assays were performed to examine the expression level of Nrf2 and the subcellular localization of Nrf2 and Drp1. Mitochondrial function was measured by the JC-1 probe and MitoSOX Red. Mitochondrial morphology was visualized by MitoTracker staining and transmission electron microscopy (TEM). After subendplate injection of the engineered exosomes, the degree of CEP degeneration and IVDD was validated radiologically and histologically.ResultsWe found that the cargo delivery efficiency of exosomes after cargo packaging was increased by surface modification. CAP-Nrf2-Exos facilitated chondrocyte-targeted delivery of Nrf2 and activated the endogenous antioxidant defence system in CEP cells. The engineered exosomes inhibited Drp1 S616 phosphorylation and mitochondrial translocation, thereby preventing mitochondrial fragmentation and dysfunction. LPS-induced CEP cell apoptosis was alleviated by CAP-Nrf2-Exo treatment. In a rat model of CEP degeneration, the engineered exosomes successfully attenuated CEP degeneration and IVDD and exhibited better repair capacity than natural exosomes.ConclusionCollectively, our findings showed that exosome-mediated chondrocyte-targeted delivery of Nrf2 was an effective strategy for treating CEP degeneration.Graphic abstract CAP-Nrf2-Exos delivered Nrf2 into CEP cells and alleviated LPS-induced apoptosis by inhibiting Drp1-mediated mitochondrial fission

Activation of the Nrf-2 pathway by pinocembrin safeguards vertebral endplate chondrocytes against apoptosis and degeneration caused by oxidative stress

AimThe occurrence and progression of intervertebral disc degeneration (IDD) are significantly influenced by the cartilaginous endplate (CEP). Pinocembrin (PIN), a type of flavonoid present in propolis and botanicals, demonstrates both antioxidant and anti-inflammatory characteristics, which could potentially be utilized in management. Therefore, it is crucial to investigate how PIN protects against CEP degeneration and its mechanisms, offering valuable insights for IDD therapy. Materials and methodsTo investigate the protective impact of PIN in vivo, we created the IDD mouse model through bilateral facet joint transection. In vitro, an IDD pathological environment was mimicked by applying TBHP to treat endplate chondrocytes. Key findingsIn vivo, compared with the IDD group, the mouse in the PIN group effectively mitigates IDD progression and CEP calcification. In vitro, the activation of the Nrf-2 pathway improves the process of Parkin-mediated autophagy in mitochondria and decreases ferroptosis in chondrocytes. This enhancement promotes cell survival by addressing the imbalance of redox during pathological conditions related to IDD. Knocking down Nrf-2 with siRNA fails to provide protection to endplate chondrocytes against apoptosis and degeneration. SignificanceThe Nrf-2-mediated activation of mitochondrial autophagy and suppression of ferroptosis play a crucial role in safeguarding against oxidative stress-induced degeneration and calcification of CEP through the protective function of PIN. To sum up, this research offers detailed explanations about how PIN can protect against apoptosis and calcification in CEP, providing valuable information about the development of IDD and suggesting possible treatment approaches.

Retracted: Evaluation of Cartilaginous Endplate Degeneration Based on Magnetic Resonance Imaging.

[This retracts the article DOI: 10.1155/2021/5534227.].

Ultrashort time-to-echo MR morphology of cartilaginous endplate correlates with disc degeneration in the lumbar spine.

Using ultrashort echo time (UTE) MRI, we determined prevalence of abnormal cartilaginous endplate (CEP), and the relationship between CEP and disc degeneration in human lumbar spines. Lumbar spines from 71 cadavers (age 14-74years) were imaged at 3T using sagittal UTE and spin echo T2 map sequences. On UTE images, CEP morphology was defined as "normal" with linear high signal intensity or "abnormal" with focal signal loss and/or irregularity. On spin echo images, disc grade and T2 values of the nucleus pulposus (NP) and annulus fibrosus (AF) were determined. 547 CEPs and 284 discs were analysed. Effects of age, sex, and level on CEP morphology, disc grade, and T2 values were determined. Effects of CEP abnormality on disc grade, T2 of NP, and T2 of AF were also determined. Overall prevalence of CEP abnormality was 33% and it tended to increase with older ages (p = 0.08) and at lower spinal levels of L5 than L2 or L3 (p = 0.001). Disc grades were higher and T2 values of the NP were lower in older spines (p < 0.001) and at lower disc level of L4-5 (p < 0.05). We found significant association between CEP and disc degeneration; discs adjacent to abnormal CEPs had high grades (p < 0.01) and lower T2 values of the NP (p < 0.05). These results suggest that abnormal CEPs are frequently found, and it associates significantly with disc degeneration, suggesting an insight into pathoetiology of disc degeneration.

Activation of Nrf2 signaling by 4-octyl itaconate attenuates the cartilaginous endplate degeneration by inhibiting E3 ubiquitin ligase ZNF598

Cartilaginous endplate (CEP) degeneration is the main early manifestations of intervertebral disc degeneration (IVDD), and is closely related to the oxidative stress. Nrf2 (nuclear factor E2-related factor 2, NFE2L2) is a vital transcriptional factor of cellular antioxidant and anti-inflammatory responses. We aimed to illustrate whether the Nrf2 which was increased in expression by 4-octyl itaconate (4OI) could attenuate intervertebral disc degeneration through suppressing macrophage associated inflammation and catabolism of cartilaginous endplate. Firstly, we detected the expression of Nrf2 in human degenerative CEPs. Then, we performed in vitro, ex vivo and in vivo (a rat-tail puncture model) experiments to explore the role of 4OI in IVDD. Also, by cell co-culture experiments, we demonstrated 4OI restrained the macrophage-associated inflammatory responses. Finally, through western blotting and immunoprecipitation (IP) assay, we clarified the ZNF598-mediated ubiquitination of Nrf2. We found decreased expression of Nrf2 in human degenerative CEPs. Using a rat IVDD model(n=6), 4OI significantly ameliorated the progression of IVDD by MR images and histological analysis. Immunofluorescence results reveal that catabolism of CEPs and macrophage-associated inflammation are suppressed by 4OI treatment. Mechanistically, the 4OI increases Nrf2 expression and inhibits the secretion of inflammatory factors (IL-1β) by Lipopolysaccharide (LPS)-induced macrophages, thus preventing the inflammatory-related CEP degeneration. Meanwhile, 4OI suppresses the reactive oxygen species (ROS) production and catabolism of LPS-induced rat CEP cells. In addition, 4OI inhibits the ZNF598-dependent ubiquitination of Nrf2 in LPS-induced rat CEP cells. 4OI may alleviate IVDD by suppressing CEP degeneration and macrophage-associated inflammation. 4OI may be an alternative therapy for degenerative CEPs/IVDs.

Identification of Differentially Expressed circRNAs, miRNAs, and Genes in Patients Associated with Cartilaginous Endplate Degeneration.

Background Intervertebral disc degeneration (IDD) disease is a global challenge because of its predominant pathogenic factor in triggering low back pain, whereas cartilaginous endplate degeneration (CEPD) is the main cause of IDD. Accumulating evidence have indicated that the differentially expressed microRNAs (DEMs) and differentially expressed genes (DEGs) have been determined to be involved in multiple biological processes to mediate CEPD progression. However, the differentially expressed circular RNAs (DECs) and their potential biofunctions in CEPD have not been identified. Methods GSE153761 dataset was analyzed using R software to predict DECs, DEMs, and DEGs. Pathway enrichment analysis of DEGs and host genes of DECs and protein-protein interaction network of DEGs were conducted to explore their potential biofunctions. Furthermore, we explore the potential relationship between DEGs and DECs. Results There were 74 DECs, 17 DEMs, and 68 DEGs upregulated whereas 50 DECs, 16 DEMs, and 67 DEGs downregulated in CEPD group. Pathway analysis unveiled that these RNAs might regulate CEPD via mediating inflammatory response, ECM metabolism, chondrocytes apoptosis, and chondrocytes growth. A total of 17 overlapping genes were predicted between the host genes of DEGs and DECs, such as SDC1 and MAOA. Moreover, 6 upregulated DECs, of which hsa_circ_0052830 was the most upregulated circRNA in CEPD, were derived from the host genes SDC1, whereas 8 downregulated DECs were derived from the host genes MAOA. Conclusion This will provide novel clues for future experimental studies to elucidate the pathomechanism of CEPD and therapeutic targets for CEPD-related diseases.

Evaluation of Cartilaginous Endplate Degeneration Based on Magnetic Resonance Imaging.

In order to carry out the evaluation of cartilaginous endplate degeneration based on magnetic resonance imaging (MRI), this paper retrospectively analyzed the MRI data from 120 cases of patients who were diagnosed as lumbar intervertebral disc degeneration and underwent MRI examinations in the designated hospital of this study from June 2018 to June 2020. All cases underwent conventional sagittal and transverse T1WI and T2WI scans, and some cases were added with sagittal fat-suppression T2WI scans; then, the number of degenerative cartilaginous endplates and its ratio to degenerative lumbar intervertebral discs were counted and calculated, and the T1WI and T2WI signal characteristics of each degenerative cartilage endplate and its correlation with cartilaginous endplate degeneration were summarized, compared, and analyzed to evaluate the cartilaginous endplate degeneration by those magnetic resonance information. The study results show that there were 33 cases of cartilaginous endplate degeneration, accounting for 27.50% of all those 120 patients with lumbar intervertebral disc degeneration (54 degenerative endplates in total), including 9 cases with low T1WI and high T2WI signals, 5 cases with high T1WI and low T2WI signals, 12 cases with high and low mixed T1WI and high or mixed T2WI signals, and 4 cases with both low T1WI and T2WI signals. Therefore, MRI scanning can clearly present the abnormal signals of lumbar intervertebral disc and cartilaginous endplate degeneration, accurately identity their lesion locations, and type their degenerative characteristics, which may be best inspection method for the evaluation of cartilaginous endplate degeneration in the early diagnosis of intervertebral disc degeneration. The study results of this paper provide a reference for further researches on the evaluation of cartilaginous endplate degeneration based on magnetic resonance imaging.

Chlorogenic Acid retards cartilaginous endplate degeneration and ameliorates intervertebral disc degeneration via suppressing NF-κB signaling

AimsIntervertebral Disc Degeneration (IDD) is a key factor involved in low back pain (LBP) which affects approximately 540 million individuals worldwide. Chlorogenic Acid (CGA), a natural compound, exerts anti-inflammatory property in several diseases. Here, we aim to investigate the biological effect of CGA on IDD and explore the underlying mechanism. Materials and methodsLumbar spine instability (LSI) model in mice was utilized to mimic process of IDD. The effects of CGA in response to LSI were evaluated by luminescent imaging, micro-CT, histomorphology, and immunohistochemistry in vivo. Besides, the cytotoxicity of CGA on chondrocytes was detected by cell counting kit-8 (CCK-8) and the biological effects were assessed by polymerase chain reaction (PCR) in vitro. Key findingsWe found that CGA treatment dramatically suppressed the NF-κB activity in LSI mice. Moreover, administration of CGA mitigated cartilaginous endplate degeneration and postponed IDD development accompanying a decrease of inflammatory and catabolic mediators. Specifically, CGA ameliorated endplate degeneration might be related to its protective effects against endplate chondrocytes apoptosis and trans-differentiation. We further elucidated that CGA exerted these biological effects mainly by repressing NF-κB signaling in cartilage endplate. SignificanceOur study has illustrated, for the first time, the curative effects as well as the latent mechanism of CGA in IDD and our results suggested that CGA administration might be used as an alternative therapy for IDD.

Comprehensive evaluation of differential long non-coding RNA and gene expression in patients with cartilaginous endplate degeneration of cervical vertebra.

Long non-coding RNAs (lncRNAs) are emerging as key regulators in gene expression; however, little is currently known regarding their role in cartilaginous endplate (CE) degeneration (CED) of cervical vertebra. The present study aimed to investigate the expression levels of lncRNAs and analyze their potential functions in CED of cervical vertebra in patients with cervical fracture and cervical spondylosis. Human competitive endogenous RNA (ceRNA) array was used to analyze lncRNA and mRNA expression levels in CE samples from patients with cervical fracture and cervical spondylosis, who received anterior cervical discectomy and fusion. Differentially expressed lncRNAs (DELs) or differentially expressed genes (DEGs) were identified and functionally analyzed, using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. An lncRNA-microRNA(miRNA)-mRNA ceRNA regulatory network was constructed based on the DELs and DEGs, and the ceRNA network was visualized using Cytoscape 3.7.2 software. In total, one downregulated mRNA, one upregulated miRNA and five downstream regulated lncRNAs were identified using reverse transcription-quantitative PCR in CED and healthy CE samples. A total of 369 lncRNAs and 246 mRNAs were identified as differentially expressed in CE. The GO and KEGG analyses demonstrated that the majority of GO and KEGG enrichments were associated with CED. Furthermore, a ceRNA network was established, including 168 putative miRNA response elements, 189 upregulated and 37 downregulated lncRNAs and 47 upregulated and 10dow regulated DEGs. The present study analyzed the function of DEGs in the ceRNA network and filtered out the same items as in DEG-function enrichment analysis. These results provide a new perspective for an improved understanding of ceRNA-mediated gene regulation in cervical spondylosis, and provide a novel theoretical basis for further studies on the function of lncRNA in cervical spondylosis. However, further experiments are required to validate the results of the present study.

Propionibacterium acnes induces cartilaginous endplate degeneration by promoting MIF expression via the NF-\u03baB pathway

BackgroundPropionibacterium acnes (P. acnes) is a novel pathogenic factor that contributes to cartilaginous endplate (CEP) degeneration. However, the underlying mechanism of P. acnes-induced CEP degeneration remains unclear. The objective of this study is to investigate the underlying mechanism of P. acnes-induced CEP degeneration.MethodsWe first examined MIF expression in degenerated human CEP samples by immunohistochemistry. We developed a P. acnes-induced rat model and detected MIF expression using immunohistochemistry. Additionally, we investigated the mechanism of P. acnes-induced CEP degeneration in CEP cells using western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR).ResultsWe found that compared with the normal human CEP, the expression of MIF was increased in the degenerated human CEP. In a rat model, P. acnes induced CEP degeneration and upregulated MIF expression significantly. More importantly, we revealed the underlying mechanism of P. acnes-induced CEP degeneration in the rat CEP cells. Firstly, P. acnes induced the expression of MIF in a concentration-dependent manner. Then, MIF upregulated the expression of MMP-13 and promoted the secretion of IL-6 and IL-1β. Finally, P. acnes may promote MIF expression via NF-κB pathway rather than ERK1/2 pathway.ConclusionP. acnes-induced MIF expression via NF-κB pathway may be the underlying mechanism of CEP degeneration.

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

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.

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.

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.

Lycorine Suppresses Endplate-Chondrocyte Degeneration and Prevents Intervertebral Disc Degeneration by Inhibiting NF-κB Signalling Pathway

Background/Aims: Cartilaginous endplate (CEP) degeneration is an important cause for intervertebral disc (IVD) degeneration that leads to low-back pain. The identification of compounds that may prevent CEP degeneration is of interest for the prevention of IVD degeneration. Methods: Catabolic protease expression in the CEP of disc degeneration patients was first assessed. The toxicity, function and underlying mechanism of lycorine (LY) on CEP-derived chondrocytes degeneration were assessed in vitro by flow cytometry analysis and western blotting. The concentration and function of LY in rat-tail disc-degeneration models were also assessed by HPLC (High Performance Liquid Chromatography) quantification and histological analysis. Results: In CEP cells, Interleukin (IL)-1β upregulated the expression of matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 that is critical for the degradation of cartilage extracellular matrix. Interestingly, LY suppressed the expression of these enzymes via the inhibition of nuclear factor-κB (NFκB) signalling and thus prevented IL-1β-induced endplate cell degeneration in vitro. More importantly, LY also reduced the expression of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 in CEP and exerted a protective effect on both CEP and nucleus pulposus (NP) degeneration. In addition to its inhibitory effect on matrix-degrading protease expression, LY treatment also reduced positive regulators of proinflammatory cytokines, such as MIF, which can be secreted by CEP cells and subsequently target NP cells. Conclusion: LY could serve as a potential drug for treating IVD disease.

Human cartilaginous endplate degeneration is induced by calcium and the extracellular calcium-sensing receptor in the intervertebral disc.

The cartilaginous endplates (CEPs) are thin layers of hyaline cartilage found adjacent to intervertebral discs (IVDs). In addition to providing structural support, CEPs regulate nutrient and metabolic exchange in the disc. In IVD pathogenesis, CEP undergoes degeneration and calcification, compromising nutrient availability and disc cell metabolism. The mechanism(s) underlying the biochemical changes of CEP in disc degeneration are currently unknown. Since calcification is often observed in later stages of IVD degeneration, we hypothesised that elevations in free calcium (Ca2+) impair CEP homeostasis. Indeed, our results demonstrated that the Ca2+ content was consistently higher in human CEP tissue with grade of disc degeneration. Increasing the levels of Ca2+ resulted in decreases in the secretion and accumulation of collagens type I, II and proteoglycan in cultured human CEP cells. Ca2+ exerted its effects on CEP matrix protein synthesis through activation of the extracellular calcium-sensing receptor (CaSR); however, aggrecan content was also affected independent of CaSR activation as increases in Ca2+ directly enhanced the activity of aggrecanases. Finally, supplementing Ca2+ in our IVD organ cultures was sufficient to induce degeneration and increase the mineralisation of CEP, and decrease the diffusion of glucose into the disc. Thus, any attempt to induce anabolic repair of the disc without addressing Ca2+ may be impaired, as the increased metabolic demand of IVD cells would be compromised by decreases in the permeability of the CEP.

The NLRP3/Caspase-1/Interleukin-1β Axis Is Active in Human Lumbar Cartilaginous Endplate Degeneration.

Modic changes are the MRI signal changes of degenerative lumbar vertebral endplate and which lead to or accelerate intervertebral disc degeneration. NLRP3, caspase-1, and interleukin-1β (IL-1β) play a pivotal role in the pathogenesis of many inflammatory diseases, such as osteoarthritis. However, the roles of IL-1β and its activators caspase-1 and NLRP3 are unclear in the degenerative endplate. We asked: (1) What are the degenerative changes of the histologic features and chondrogenic markers' gene expressions between the cartilaginous endplates of patients with Modic changes and trauma (control)? (2) How does the NLRP3/caspase-1/IL-1β axis in the cartilaginous endplates of patients with Modic changes compare with control (trauma) specimens? Surgical specimens of cartilaginous endplates were divided into Modic changes (n = 56) and the trauma control (n = 16) groups. Hematoxylin and eosin and safranin O staining of cartilaginous endplate tissues were done to evaluate the extracellular matrix. Reverse transcription-polymerase chain reaction was performed on these tissues to investigate mRNA expression of type II collagen (Col II), SOX-9, matrix metalloproteinase-3, and a disintegrin like and metalloproteinase thrombospondin type I motifs-5. NLRP3, caspase-1, and IL-1β were evaluated by reverse transcription-polymerase chain reaction and immunohistochemistry. Hematoxylin and eosin and safranin O staining showed the extracellular matrix degraded in the cartilaginous endplates of patients with Modic changes but not in the control cartilaginous endplates. Chondrogenic Col II (p = 0.024) and SOX9 (p = 0.053) were downregulated in the Modic changes group compared with the control group. In contrast to the control group, the transcriptional levels of NLRP3 (p < 0.001), caspase-1 (p = 0.054), and IL-1β (p = 0.001) were all upregulated in the Modic changes group. The expression of NLRP3, caspase-1, and IL-1β was upregulated in the patients with low back pain and Modic changes on MRI compared with patients with vertebral burst fracture without degenerative changes on MRI. The data suggest the NLRP3/caspase-1/IL-1β axis may be implicated in lumbar cartilaginous endplate degeneration. The NLRP3/caspase-1/IL-1β axis is active in cartilaginous endplates of patients with Modic changes and inflammatory cascades can exacerbate the cartilaginous endplate degeneration which may act as a trigger for intervertebral disc degeneration and low back pain. If these findings can be confirmed by others, we hope that new and effective therapy could be developed directed against this target.

The Extracellular Calcium-Sensing Receptor as a Contributor in Human Cartilaginous Endplate Degeneration

Introduction Intervertebral discs (IVDs) are virtually avascular tissue relying on the diffusion of nutrients and metabolites from blood vessels in the outer annulus fibrosus, and in the vertebral bodies that partially penetrate the subchondral plate, and terminate at the cartilaginous endplate (CEP). Degeneration of CEP is viewed as a catalyst to IVD degeneration, as it impedes nutrient diffusion to the disc. Similar to articular cartilage, degeneration of CEP involves decreased proteoglycan and type II collagen (Col II) content, thinning, increased type X collagen (Col X), and advanced calcification. Although biological repair strategies of the degenerative disc involving supplementation of growth factors are being pursued, disc nutrition is an important factor in considering their success in disc repair. The extracellular calcium-sensing receptor (CaSR) is a G protein-coupled receptor, and currently, is the only known mechanism in sensing both local and systemic calcium fluctuations. CaSR is the principle regulator of parathyroid hormone synthesis and secretion from the parathyroid gland, and in addition to its expression in other calcium-handling tissues (intestine, kidney), evidence suggests a direct involvement in bone and cartilage development. Recently, CaSR has been shown to modulate vascular calcification; however, its function in the calcification of other tissues remains unknown. Objectives Calcification of CEP is suggested to be a catalyst in the degeneration of IVDs. However, the mechanism(s) of CEP degeneration are currently unknown. We propose that CaSR activation modulates the degeneration of CEP, by increasing the expression of catabolic factors in CEP chondrocytes, resulting from the accumulation of ionic calcium in the milieu. Materials and Methods Human CEPs were obtained from donor IVDs of Thompson grades 2, 3, and 4 through organ donations within 24 hours of death. CEP chondrocytes 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 CEP tissue to validate the grade and expression of CaSR. Free calcium levels were also measured and compared between grades. Immunocytochemistry, Western blotting, and MAPK signaling were performed on cultured CEP chondrocytes of various grades to demonstrate expression of CaSR and degenerative markers, including the function of the receptor. CEP cells of various grades were cultured in medium containing either high calcium (5 mM Ca2+), CaSR allosteric agonist (cinacalcet, 1 µM), or regular growth medium (control, 1 mM Ca2+) for 7 days, and monitored for changes in markers of degeneration and CaSR expression. Results Ionic calcium content in CEP tissue increased significantly with IVD degeneration (Fig. A). Interestingly, immunohistochemistry on CEP tissue also revealed an increase in the expression of CaSR that paralleled the degree of IVD degeneration. Immunocytochemistry and Western blotting on isolated CEP cells demonstrated an upregulation of CaSR (Fig. B) and type X collagen that varied with Thompson grades. The expression of matrix metalloproteinase (MMP)-13 was also increased with degeneration; however, the expression of Col II and the proteoglycan aggrecan (Agg) were inversely related to Thompson grade. The magnitude of CaSR signaling in CEP cells when stimulated with 5 mM Ca2+, as determined by ERK phosphorylation, was significantly enhanced in grade 4 versus grade 2 CEP chondrocytes. To determine a role of CaSR in the degeneration of CEP, we cultured Thompson grade 2 CEP chondrocytes in 1 mM Ca2+ (control medium), 5 mM Ca2+ or cinacalcet (allosteric CaSR agonist, 1 µM) for 7 days. Western blot analysis on lysate from G2 CEP cells treated with 5 mM Ca2+ or cinacalcet demonstrated significant increases in the expression of Col X and MMP-13 (Figs. D and E), however, decreased expression of Col II and Agg was observed (Fig. C). Conclusion CaSR has been described as a mediator in ectopic calcification, and its expression is upregulated in CEP of degenerative discs. We provide evidence that CaSR upregulation may be a hallmark of CEP calcification. Activation of CaSR either by its agonist, calcium or cinacalcet, lead to increases in the synthesis of degenerative factors (Col X and MMP-13), and decreases in the production of matrix proteins (Col II and Agg). An understanding on the mechanism(s) of CEP degeneration is key when considering growth factor substitution in IVD repair, as disc nutrition may be an important factor in determining their success. Disclosure of Interest None declared References Lotz JC, Fields AJ, Liebenberg EC. The role of the vertebral end plate in low back pain. Global Spine J 2013;3(3):153–164 Brown EM. Role of the calcium-sensing receptor in extracellular calcium homeostasis. Best Pract Res Clin Endocrinol Metab 2013;27(3):333–343