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Background: Intervertebral disc (IVD) degeneration is a multifactorial pathological process resulting in the dysregulation of IVD cell activity. The catabolic shift observed in IVD cells during degeneration leads to increased inflammation, extracellular matrix (ECM) degradation, aberrant intracellular signaling and cell loss. Importantly, these pathological processes are known to be interconnected and to collectively contribute to the progression of the disease. MicroRNAs (miRNAs) are known as strong post-transcriptional regulators, targeting multiple genes simultaneously and regulating numerous intracellular pathways. Specifically, miR-155-5p has been of particular interest since it is known as a pro-inflammatory mediator and contributing factor to diseases like cancer and osteoarthritis. This study investigated the role of miR-155-5p in IVD degeneration with a specific focus on inflammation and mechanosensing., Methods: Gain- and loss-of-function studies were performed through transfection of human Nucleus pulposus (NP) and Annulus fibrosus (AF) cells isolated from degenerated IVDs with miR-155-5p mimics, inhibitors or their corresponding non-targeting control. Transfected cells were then subjected to an inflammatory environment or mechanical loading. Conditioned media and cell lysates were collected for phosphorylation and cytokine secretion arrays as well as gene expression analysis., Results: Increased expression of miR-155-5p in AF cells resulted in significant upregulation of interleukin (IL)-8 cytokine secretion during cyclic stretching and a similar trend in IL-6 secretion during inflammation. Furthermore, miR-155-5p mimics increased the expression of the brain-derived neurotrophic factor (BDNF) in AF cells undergoing cyclic stretching. In NP cells, miR-155-5p gain-of-function resulted in the activation of the mitogen-activated protein kinase (MAPK) signaling pathway through increased phosphorylation of p38 and p53. Lastly, miR-155-5p inhibition caused a significant increase in the anti-inflammatory cytokine IL-10 in AF cells and the tissue inhibitor of metalloproteinases (TIMP)-4 in NP cells respectively., Conclusion: Overall, these results show that miR-155-5p contributes to IVD degeneration by enhancing inflammation through pro-inflammatory cytokines and MAPK signaling, as well as by promoting the catabolic shift of AF cells during mechanical loading. The inhibition of miR-155-5p may constitute a potential therapeutic approach for IVD degeneration and low back pain., (© 2024. The Author(s).)

Syndecan 4 (SDC4), a cell surface heparan sulfate proteoglycan, is known to regulate matrix catabolism by nucleus pulposus cells in an inflammatory milieu. However, the role of SDC4 in the aging spine has never been explored. Here we analyzed the spinal phenotype of Sdc4 global knockout (KO) mice as a function of age. Micro-computed tomography showed that Sdc4 deletion severely reduced vertebral trabecular and cortical bone mass, and biomechanical properties of vertebrae were significantly altered in Sdc4 KO mice. These changes in vertebral bone were likely due to elevated osteoclastic activity. The histological assessment showed subtle phenotypic changes in the intervertebral disc. Imaging-Fourier transform-infrared analyses showed a reduced relative ratio of mature collagen crosslinks in young adult nucleus pulposus (NP) and annulus fibrosus (AF) of KO compared to wildtype discs. Additionally, relative chondroitin sulfate levels increased in the NP compartment of the KO mice. Transcriptomic analysis of NP tissue using CompBio, an AI-based tool showed biological themes associated with prominent dysregulation of heparan sulfate GAG degradation, mitochondria metabolism, autophagy, endoplasmic reticulum (ER)-associated misfolded protein processes and ER to Golgi protein processing. Overall, this study highlights the important role of SDC4 in fine-tuning vertebral bone homeostasis and extracellular matrix homeostasis in the mouse intervertebral disc., Competing Interests: Declaration of competing interests Authors of this manuscript do not have conflicts of interest to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The objective of this research was to identify differentially expressed genes (DEGs) related to ferroptosis in the annulus fibrosus (AF) during intervertebral disc degeneration (IDD)., Methods: We analyzed gene data from degenerated and normal AF obtained from the GSE70362 and GSE147383 datasets. An analysis to determine the functional significance of the DEGs was conducted, followed by the creation of a network illustrating the interactions between proteins. We further analyzed the immune infiltration of the DEGs and determined the hub DEGs using LASSO regression analysis. Finally, we identified the hub ferroptosis-related DEGs (FRDEGs) and verified their expression levels using Real-time quantitative polymerase chain reaction (RT-qPCR), Western blot, Immunohistochemical Staining (IHC), and Immunofluorescence (IF)., Results: By analyzing the GSE70362 and GSE147383 datasets, we identified 118 DEGs. In degenerative AF groups, we observed a significant increase in immune infiltration of resting memory CD4+ T cells. LASSO regression analysis revealed 9 hub DEGs. The construction of a Receiver Operating Characteristic (ROC) curve yielded an Area Under the Curve (AUC) value of 0.762. Furthermore, we found that MGST1 is a hub gene related to ferroptosis. Our examination of immune infiltration indicated that MGST1 primarily influences macrophage M0 in different immune cell expression groups. Finally, our observations revealed a marked upregulation of MGST1 expression in the degenerated annulus fibrosus tissue., Conclusion: Our findings indicate an upsurge in MGST1 levels within degenerative AF, potentially playing a crucial role in the exacerbation of IDD. These findings provide a foundation for further exploration of the pathological mechanisms underlying IDD and offer potential drug targets for intervention., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

Intervertebral disc herniation is a common spinal disorder that is often treated with discectomy when conservative measures fail. To devise therapeutic strategies for tears in the annulus fibrosus (AF), the regenerative capability of AF cells under spinal loading needs to be addressed. We hypothesized that the compressive loading associated with deformation in AF cells reduces synthetic and degradative activities in extracellular matrix and cell proliferation. We evaluated expression of key matrix molecules and cell proliferation by RT-PCR and immunohistochemistry by inner and outer bovine AF cells incubated under hydrostatic pressure (HP), arc-bending strain (Strain), and combined HP and Strain (HP/Strain) mimicking spinal loading. Inner AF cells showed significantly increased levels of aggrecan core protein, chondroitin sulfate N-acetylgalactosaminyltransferase-1, and tissue inhibitor of metalloproteinases-2 by 6 days under HP (p < 0.05), with a tendency toward increased matrix metalloproteinase-13. Outer AF cells demonstrated a significant decline in collagen type-2 under Strain and HP/Strain (p < 0.05) and a tendency toward suppression of collagen type-1 and elastin expression compared to HP and unloaded control. On the other hand, proliferating cell nucleus antigen increased significantly under Strain and HP/Strain in inner AF and declined under unloaded and HP in outer AF (p < 0.05). Immunohistology findings supported reductions in gene expressions of matrix molecules. Thus, changes in HP/Strain in AF appear to diminish synthetic and degradative activities while increasing cell proliferation. To promote regeneration, continuous overloading should be avoided, as it converts the synthetic activity to a state in which tissue repair is limited., (© 2024 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Low back pain stands as a significant factor in disability, largely resulting from intervertebral disc degeneration (IVDD). High glucose (HG) levels have been implicated in the pathogenesis of IVDD. However, the detailed mechanism of HG in IVDD is largely unknown. Our clinical results revealed that fibrosis markers such as CTGF, Col1a1, ATF4, and EIF2 are highly expressed in advanced-stage IVDD patients. Stimulation of human annulus fibrosus cells (HAFCs) with HG, but not mannitol, promotes fibrosis protein production. Ingenuity Pathway Analysis in the GSE database found that the mTOR, PKCδ, and NF-κB pathways were significantly changed during IVDD. The mTOR, PKCδ, and NF-κB inhibitors or siRNAs all abolished HG-induced fibrosis protein production. In addition, treatment of HAFCs with HG enhances the activation of mTOR, PKCδ, and NF-κB pathways. Thus, HG facilitates fibrosis in IVDD through mTOR, PKCδ, and NF-κB pathways. These results underscore the critical role of HG as a fibrotic factor in the progression of IVDD.

Elucidating how cell populations promote onset and progression of intervertebral disc degeneration (IDD) has the potential to enable more precise therapeutic targeting of cells and mechanisms. Single-cell RNA-sequencing (scRNA-seq) is performed on surgically separated annulus fibrosus (AF) (19,978; 26,983 cells) and nucleus pulposus (NP) (20,884; 24,489 cells) from healthy and diseased human intervertebral discs (IVD). In both tissue types, depletion of cell subsets involved in maintenance of healthy IVD is observed, specifically the immature cell subsets - fibroblast progenitors and stem cells - indicative of an impairment of normal tissue self-renewal. Tissue-specific changes are also identified. In NP, several fibrotic populations are increased in degenerated IVD, indicating tissue-remodeling. In degenerated AF, a novel disease-associated subset is identified, which expresses disease-promoting genes. It is associated with pathogenic biological processes and the main gene regulatory networks include thrombospondin signaling and FOXO1 transcription factor. In NP and AF cells thrombospondin protein promoted expression of genes associated with TGFβ/fibrosis signaling, angiogenesis, and nervous system development. The data reveal new insights of both shared and tissue-specific changes in specific cell populations in AF and NP during IVD degeneration. These identified mechanisms and molecules are novel and more precise targets for IDD prevention and treatment., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Purpose: This study explores the potential of Omilancor in treating Intervertebral Disc Degeneration (IDD) through MAP2K6 targeting., Methods: We analyzed mRNA microarray datasets to pinpoint MAP2K6 as a key regulator implicated in IDD progression. Follow-up studies demonstrated that cisplatin (DDP) could prompt cellular senescence in vitro by upregulating MAP2K6 expression. Through molecular docking and other analyses, we identified Omilancor as a compound capable of binding to MAP2K6. This interaction effectively impeded the cellular senescence induced by DDP., Results: We further showed that administration of Omilancor could significantly alleviate the degeneration of IVDs in annulus fibrosus puncture-induced rat model., Conclusions: Omilancor shows promise as a treatment for IDD by targeting MAP2K6-mediated cellular senescence.

Intervertebral disc degeneration (IVDD) has been considered a major cause of low back pain. Therefore, further molecular subtypes of IVDD and identification of potential critical genes are urgently needed. First, consensus clustering was used to classify patients with IVDD into two subtypes and key module genes for subtyping were identified using weighted gene co-expression network analysis (WGCNA). Then, key module genes for the disease were identified by WGCNA. Subsequently, SVM and GLM were used to identify hub genes. Based on the above genes, a nomogram was constructed to predict the subtypes of IVDD. Finally, we find that ROM1 is lowered in IVDD and is linked to various cancer prognoses. The present work offers innovative diagnostic and therapeutic biomarkers for molecular subtypes of IVDD.

Background Context: In clinical practice, acute trauma and chronic degeneration of the annulus fibrosus (AF) can promote further degeneration of the intervertebral disc (IVD). Therefore, it is critical to understand the AF repair process and its consequences on IVD. However, the lack of cost-effective and reproducible in vivo animal models of AF injury has limited research development in this field., Purposes: The purpose of this study was to establish and evaluate the utility of a novel animal model for full-thickness AF injury. Three foci were proposed: (1) whether this new modeling method can cause full-layer AF damage; (2) the repair processes and pathological changes in the damaged area after AF injury, and (3) the morphological and histological changes in the IVD are after AF injury., Study Design/setting: In vivo rat AF injury model with characterization of AF damage repair, IVD degeneration., Methods: A total of 72,300 g male rats were randomly assigned to one of the two groups: experimental or sham. Annulus fibrosus was separated layer by layer under the microscope with a #11 blade up to the AF- nucleus pulpous (NP) junction. The repair process of the horizontal AF and morphological changes in the sagittal IVD were evaluated with HE staining. Sirius red staining under polarized light. Immunofluorescence was conducted to analyze changes in the expression of COL1 and COL3 in the AF injury area and 8-OHdg, IL-6, MMP13, FSP1, and ACAN in the IVD. The disc height and structural changes after AF injury were measured using X-ray and contrast-enhanced micro-CT. Additionally, the resistance of the AF to stretching was analyzed using three-point bending., Results: Annulus fibrosus-nucleus pulpous border was identified to stably induce the full-thickness AF injury without causing immediate NP injury. The AF repair process after injury was slow and expressed inflammation factors continuously, with abundant amounts of type III collagen appearing in the inner part of the AF. The scar at the AF lesion had decreased resistance to small molecule penetration and weakened tensile strength. Full-thickness AF injury induced disc degeneration with loss of disc height, progressive unilateral vertebral collapse, and ossification of the subchondral bone. Inflammatory-induced degeneration and extracellular matrix catabolism gradually appeared in the NP and cartilage endplate (CEP)., Conclusions: We established a low-cost and reproducible small animal model of AF injury which accurately replicated the pathological state of the limited AF self-repair ability and demonstrated that injury to the AF alone could cause further degeneration of the IVD., Clinical Relevance: This in vivo rat model can be used to study the repair process of the AF defect and pathological changes in the gradual degeneration of IVD after AF damage. In addition, the model provides an experimental platform for in vivo experimental research of potential clinical therapeutics., Competing Interests: Declaration of Competing Interest One or more of the authors declare financial or professional relationships on ICMJE-TSJ disclosure forms., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Low back pain is a common chronic disease that can severely affect the patient's work and daily life. The breakdown of spinal mechanical homeostasis caused by intervertebral disc (IVD) degeneration is a leading cause of low back pain. Annulus fibrosus (AF), as the outer layer structure of the IVD, is often the first affected part. AF injury caused by consistent stress overload will further accelerate IVD degeneration. Therefore, regulating AF injury repair and remodeling should be the primary goal of the IVD repair strategy. Mechanical stimulation has been shown to promote AF regeneration and repair, but most studies only focus on the effect of single stress on AF, and lack realistic models and methods that can mimic the actual mechanical environment of AF. In this article, we review the effects of different types of stress stimulation on AF injury repair and remodeling, suggest possible beneficial load combinations, and explore the underlying molecular mechanisms. It will provide the theoretical basis for designing better tissue engineering therapy using mechanical factors to regulate AF injury repair and remodeling in the future.

Background: Intervertebral disc degeneration (IVDD) is considered one of the main reasons for low back pain (LBP). To date, the specific pathology of IVDD remains unclear. The annulus fibrosus (AF) is an important part of the intervertebral disc, and AF cell oxidative stress, apoptosis plays a vital role in disc degeneration. Protein phosphatase 2 A (PP2A), a serine/threonine phosphatase, has regulatory functions in various processes, including apoptosis and autophagy. However, thus far, the effect of PP2A on IVDD is not clear., Methods: AF cells derived from caudal intervertebral discs in SD rats were used to analyze the levels of oxidative stress, apoptosis and degeneration as well as PP2A expression. A PP2A agonist (FTY720), inhibitor (microcystin-LR) and siRNA (si-PPP2CA) were employed in IVDD induced by H 2 O 2 to investigate the levels of apoptosis and degeneration. The p38/MAPK signal pathways were evaluated, and a p38 inhibitor (SB203580) and ERK inhibitor (U0126) were added for verification. Finally, FTY720 and microcystin-LR were administered to IVDD rats to assess the effects on levels of apoptosis and degeneration and the relief of IVDD., Results: The expression of PP2A was increased in rat AF cells after H 2 O 2 intervention. The levels of apoptosis and degeneration were higher with upregulation of PP2A but were significantly reduced after inhibition of PP2A. The PP2A inhibitor relieved cell apoptosis and degeneration by downregulating the p38/MAPK pathway. In vivo, the knockdown of PP2A resulted in a more complete morphology of discs and less apoptotic and degenerative expression., Conclusions: This study suggests that the downregulation of PP2 A could reduce AF cell apoptosis and degeneration via the p38/MAPK pathway. It also revealed that the inhibition of PP2 A is expected to be a therapeutic target for IVDD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Intervertebral disc degeneration (IDD) is a leading cause of low back pain. The inflammatory responses caused by aberrant mechanical loading are one of the major factors leading to annulus fibrosus (AF) degeneration and IDD. Previous studies have suggested that moderate cyclic tensile strain (CTS) can regulate anti-inflammatory activities of AF cells (AFCs), and Yes-associated protein (YAP) as a mechanosensitive coactivator senses diverse types of biomechanical stimuli and translates them into biochemical signals controlling cell behaviors. However, it remains poorly understood whether and how YAP mediates the effect of mechanical stimuli on AFCs. In this study, we aimed to investigate the exact effects of different CTS on AFCs as well as the role of YAP signaling involving in it. Our results found that 5% CTS inhibited the inflammatory response and promoted cell growth through inhibiting the phosphorylation of YAP and nuclear localization of NF-κB, while 12% CTS had a significant proinflammatory effect with the inactivation of YAP activity and the activation of NF-κB signaling in AFCs. Furthermore, moderate mechanical stimulation may alleviate the inflammatory reaction of intervertebral discs through YAP-mediated suppression of NF-κB signaling in vivo. Therefore, moderate mechanical stimulation may serve as a promising therapeutic approach for the prevention and treatment of IDD., (© 2023 Orthopaedic Research Society.)

Background Context: Intervertebral disc degeneration (IVDD) is an incurable, specific treatment-orphan disease with an increasing burden worldwide. Although great efforts have been made to develop new regenerative therapies, their clinical success is limited., Purpose: Characterize the metabolomic and gene expression changes underpinning human disc degeneration. This study also aimed to disclose new molecular targets for developing and optimizing novel biological approaches for IVDD., Study Design: Intervertebral disc cells were obtained from IVDD patients undergoing circumferential arthrodesis surgery or from healthy subjects. Mimicking the harmful microenvironment of degenerated discs, cells isolated from the nucleus pulposus (NP) and annulus fibrosus (AF) were exposed to the proinflammatory cytokine IL-1β and the adipokine leptin. The metabolomic signature and molecular profile of human disc cells were unraveled for the first time., Methods: The metabolomic and lipidomic profiles of IVDD and healthy disc cells were analyzed by high-performance liquid chromatography-mass spectrometry (UHPLC-MS). Gene expression was investigated by SYBR green-based quantitative real-time RT-PCR. Altered metabolites and gene expression were documented., Results: Lipidomic analysis revealed decreased levels of triacylglycerols (TG), diacylglycerol (DG), fatty acids (FA), phosphatidylcholine (PC), lysophosphatidylinositols (LPI) and sphingomyelin (SM), and increased levels of bile acids (BA) and ceramides, likely promoting disc cell metabolism changing from glycolysis to fatty acid oxidation and following cell death. The gene expression profile of disc cells suggests LCN2 and LEAP2/GHRL as promising molecular therapeutic targets for disc degeneration and demonstrates the expression of genes related to inflammation (NOS2, COX2, IL-6, IL-8, IL-1β, and TNF-α) or encoding adipokines (PGRN, NAMPT, NUCB2, SERPINE2, and RARRES2), matrix metalloproteinases (MMP9 and MMP13), and vascular adhesion molecules (VCAM1)., Conclusions: Altogether, the presented results disclose the NP and AF cell biology changes from healthy to degenerated discs, allowing the identification of promising molecular therapeutic targets for intervertebral disc degeneration., Clinical Significance: Our results are relevant to improving current biological-based strategies aiming to repair IVD by restoring cellular lipid metabolites as well as adipokines homeostasis. Ultimately, our results will be valuable for successful, long-lasting relief of painful IVDD., Competing Interests: Declaration of Competing Interest One or more of the authors declare financial or professional relationships on ICMJE-TSJ disclosure forms., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Over the past decade, single-cell RNA sequencing (scRNA-seq) has revolutionized research on biological mechanisms of diseases. Moreover, this technique has been utilized to identify and characterize unique cell types and subpopulations, thereby illuminating cellular heterogeneity. The true value of scRNA-seq lies in its ability to detect transcriptional alterations or perturbed pathways within specific cell types under pathological conditions. In the context of intervertebral disc degeneration (IVDD), the pathophysiological foundation is largely rooted in inflammation. The primary target cells of IVDD are nucleus pulposus cells, annulus fibrosus cells, cartilage endplate cells, and macrophages. The advancements in scRNA-seq technology have triggered remarkable progress in IVDD treatment, leading to breakthroughs in the identification of cell subsets, functional analysis, novel therapeutic targets, and the differentiation and development of various cell types. This review is the first of its kind to introduce the application of scRNA-seq techniques in IVDD, with a focus on the most recent scRNA-seq studies that have defined the populations of various cell types and specific cell-cell interactions in IVDD. Furthermore, we highlight several promising future research directions for scRNA-seq in IVDD., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Intervertebral disc degeneration (IVDD) is closely associated with the structural damage in the annulus fibrosus (AF). Aberrant mechanical loading is an important inducement of annulus fibrosus cells (AFCs) apoptosis, which contributes to the AF structural damage and aggravates IVDD, but the underlying mechanism is still unclear. This study aims to investigate the mechanism of a mechanosensitive ion channel protein Piezo1 in aberrant mechanical loading-induced AFCs apoptosis and IVDD., Methods: Rats were subjected to lumbar instability surgery to induce the unbalanced dynamic and static forces to establish the lumbar instability model. MRI and histological staining were used to evaluate the IVDD degree. A cyclic mechanical stretch (CMS)-stimulated AFCs apoptosis model was established by a Flexcell system in vitro. Tunel staining, mitochondrial membrane potential (MMP) detection, and flow cytometry were used to evaluate the apoptosis level. The activation of Piezo1 was detected using western blot and calcium fluorescent probes. Chemical activator Yoda1, chemical inhibitor GSMTx4, and a lentiviral shRNA-Piezo1 system (Lv-Piezo1) were utilized to regulate the function of Piezo1. High-throughput RNA sequencing (RNA-seq) was used to explore the mechanism of Piezo1-induced AFCs apoptosis. The Calpain activity and the activation of Calpain2/Bax/Caspase3 axis were evaluated by the Calpain activity kit and western blot with the siRNA-mediated Calapin1 or Calpain2 knockdown. Intradiscal administration of Lv-Piezo1 was utilized to evaluate the therapeutic effect of Piezo1 silencing in IVDD rats., Results: Lumbar instability surgery promoted the expression of Piezo1 in AFCs and stimulated IVDD in rats 4 weeks after surgery. CMS elicited distinct apoptosis of AFCs, with enhanced Piezo1 activation. Yoda1 further promoted CMS-induced apoptosis of AFCs, while GSMTx4 and Lv-Piezo1 exhibited opposite effects. RNA-seq showed that knocking down Piezo1 inhibited the calcium signaling pathway. CMS enhanced Calpain activity and elevated the expression of BAX and cleaved-Caspase3. Calpain2, but not Calpain1 knockdown, inhibited the expression of BAX and cleaved-Caspase3 and alleviated AFCs apoptosis. Lv-Piezo1 significantly alleviated the progress of IVDD in rats after lumbar instability surgery., Conclusions: Aberrant mechanical loading induces AFCs apoptosis to promote IVDD by activating Piezo1 and downstream Calpain2/BAX/Caspase3 pathway. Piezo1 is expected to be a potential therapeutic target in treating IVDD., (© 2023. The Author(s).)

Intervertebral disc (IVD) degeneration is a major contributing factor for discogenic low back pain (LBP), causing a significant global disability. The IVD consists of an inner core proteoglycan-rich nucleus pulposus (NP) and outer lamellae collagen-rich annulus fibrosus (AF) and is confined by a cartilage end plate (CEP), providing structural support and shock absorption against mechanical loads. Changes to degenerative cascades in the IVD cause dysfunction and instability in the lumbar spine. Various treatments include pharmacological, rehabilitation or surgical interventions that aim to relieve pain; however, these modalities do not halt the pathologic events of disc degeneration or promote tissue regeneration. Loss of stem and progenitor markers, imbalance of the extracellular matrix (ECM), increase of inflammation, sensory hyperinnervation and vascularization, and associated signaling pathways have been identified as the onset and progression of disc degeneration. To better understand the pain originating from IVD, our review focuses on the anatomy of IVD and the pathophysiology of disc degeneration that contribute to the development of discogenic pain. We highlight the key mechanisms and associated signaling pathways underlying disc degeneration causing discogenic back pain, current clinical treatments, clinical perspective and directions of future therapies. Our review comprehensively provides a better understanding of healthy IVD and degenerative events of the IVD associated with discogenic pain, which helps to model painful disc degeneration as a therapeutic platform and to identify signaling pathways as therapeutic targets for the future treatment of discogenic pain.

Background: Intervertebral disc degeneration (IVDD) is closely associated with senescence. Annulus fibrosus (AF) cell senescence is a crucial driver of AF tissue tearing and fissures, thereby exacerbating IVDD. Increased advanced oxidative protein products (AOPPs) were found in human degenerative discs and aged rat discs and may be involved in IVDD. This study aimed to explore the mechanism of AOPPs-induced senescence in AF cells., Methods: The pathological effects of AOPPs in vivo were investigated using a rat lumbar disc persistent degeneration model and a rat caudal disc puncture model. Rat primary AF cells were selected as in vitro models, and AOPPs were used as direct stimulation to observe their pathological effects. Setanaxb (NOX1/4 inhibitor), apocynin (NADPH oxidase inhibitor) and adenovirus (ADV) packed NADPH oxidase 4 (NOX4) specific shRNAs were used for pathway inhibition, respectively. Finally, adeno-associated viruses (AAVs) packed with NOX4-specific blocking sequences were used to inhibit the in vivo pathway., Results: AOPPs accumulated in the rat lumbar and caudal degenerative discs. Intra-discal loading of AOPPs up-regulated the expression of NOX4, p53, p21, p16, IL-1 β , and TNF- α , ultimately accelerating IVDD. Exposure of AOPPs to AF primary cells up-regulated NOX4 expression, induced phosphorylation of mitogen-activated protein kinases (MAPK), triggered senescence and increased IL-1 β and TNF- α . Apocynin, setanaxib, and ADV pre-cultured AF cells abrogated AOPPs-induced senescence. AAV-mediated inhibition of NOX4 expression in vivo reduced the expression of p53, p21, p16, IL-1 β and TNF- α in vivo and delayed IVDD., Conclusions: AOPPs induced AF cell senescence through a NOX4-dependent and MAPK-mediated pathway., Competing Interests: The authors declare there are no competing interests., (©2022 Dai et al.)

Excessive mechanical loading is a major cause of spinal degeneration, typically originating from a tear in the annulus fibrosus (AF). Endoplasmic reticulum (ER) stress and NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome have been implicated in the pathogenesis of intervertebral disc (IVD) degeneration. However, the causal relationship between the mechanical stretching of AF cells and the NLRP3 inflammasome response associated with ER stress remains scarce. To elucidate the pathogenesis and regulatory mechanisms of mechanical stretch-induced IVD degeneration, human AF cell lines were subjected to different degrees of cyclic stretching to simulate daily spinal movements. Our results indicated that 15% high cyclic stretch (HCS) induced the expression of NLRP3 and interleukin-1 beta (IL-1β) and was also responsible for the increased expression of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 2 (NOX2) and reactive oxygen species (ROS) in human AF cells. In addition, HCS increased the expression of glucose-regulated protein 78 (GRP78), an ER stress chaperone, which was neutralized with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor. In addition, HCS was found to induce thioredoxin-interacting protein (TXNIP) expression and NLRP3 inflammasome activation, which can be suppressed by si-NOX2 or the NOX2 inhibitor GSK2795039. Consequently, HCS upregulated ER stress and ROS production, leading to increased NLRP3 and IL-1β expression in human AF cells, and may further accelerate IVD degeneration.

The mouse outer annulus fibrosus (AF) was previously shown to contain CD146 + AF cells, while in vitro culture and exposure to transforming growth factor-beta (TGF-β) further increased the expression of CD146. However, neither the specific function of CD146 nor the underlying mechanism of TGF-β upregulation of CD146 + AF cells have been elucidated yet. In the current study, CD146 expression and its role in cultured human AF cells was investigated studying the cells' capacity for matrix contraction and gene expression of functional AF markers. In addition, TGF-β pathways were blocked by several pathway inhibitors and short hairpin RNAs (shRNAs) targeting SMAD and non-SMAD pathways to investigate their involvement in TGF-β-induced CD146 upregulation. Results showed that knockdown of CD146 led to reduction in AF cell-mediated collagen gel contraction, downregulation of versican and smooth muscle protein 22α (SM22α), and upregulation of scleraxis. TGF-β-induced CD146 upregulation was significantly blocked by inhibition of TGF-β receptor ALK5, and partially inhibited by shRNA against SMAD2 and SMAD4 and by an Protein Kinase B (AKT) inhibitor. Interestingly, the inhibition of extracellular signal-regulated kinases (ERK) pathway induced CD146 upregulation. In conclusion, CD146 was shown to be crucial to maintain the cell contractility of human AF cells in vitro. Furthermore, TGF-β upregulated CD146 via ALK5 signaling cascade, partially through SMAD2, SMAD4, and AKT pathway, whereas, ERK was shown to be a potential negative modulator. Our findings suggest that CD146 can potentially be used as a functional marker in AF repair strategies., (© 2021 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Background: Abnormal Smad7 expression can lead to apoptosis in different cell types. Previously, we found high expression of Smad7 in rat degenerative discs. However, the exact role of Smad7 in the apoptosis of disc cells and the possible underlying mechanism remain unclear., Methods: Degenerative and nondegenerative human lumbar intervertebral discs were collected from patients during operation. The expressions of SMAD7 mRNA and protein in the different components of these discs were measured with real-time PCR and Western blotting, respectively. Annulus fibrosus (AF) cells were isolated and cultivated from the discs of young healthy rats. Smad7 in the AF cells was overexpressed with adenovirus and knocked down with siRNA. IL-1β was used to induce apoptosis in the AF cells. Loss-and-gain cell function experiments were performed to show the effect of Smad7 on the apoptosis of AF cells. The function recovery experiments were performed to verify whether Smad7 regulates the apoptosis of AF cells through the mitochondria-mediated pathway., Results: Both the mRNA and protein expressions of Smad7 were significantly higher in the different components of human degenerative discs than in those of the nondegenerative discs. IL-1β stimulated apoptosis while upregulating the Smad7 expression in the AF cells in vitro . Overexpression of Smad7 in AF cells exaggerated the IL-1β -induced apoptosis in the cells while knockdown of Smad7 expression suppressed this apoptosis. With the exaggerated apoptosis in the AF cells with Smad7 overexpression, both active cleaved caspase-3 and cleaved caspase-9, the ratio of Bax/Bcl-2, and Cyt-c increased significantly. However, the inhibitor of caspase-9, Z-LEHD-FMK, significantly diminished the apoptosis in these cells., Conclusion: Smad7 is highly expressed in human degenerative discs and participates in IL-1β-induced apoptosis of rat AF cells via the mitochondria pathway. Smad7 may be a potential target for the prevention and treatment of degenerative disc disease., Competing Interests: The authors confirm no conflicts of interest., (Copyright © 2022 Bo Li et al.)

Background Context: As no infiltrating macrophages exist in healthy discs, understanding the role of infiltrating macrophages including their polarity (M1 and M2 phenotypes) in intervertebral discs (IVDs) is important in the assessment of the pathomechanisms of disc degeneration., Purpose: To determine the relationship between infiltrating macrophage polarization and the progression of human cervical IVD degeneration., Study Design: Histopathological study using harvested human cervical IVDs., Methods: IVDs collected during anterior cervical decompression from 60 patients were subjected to immunostaining and immunoblotting. The samples were classified as type 0-3 according to the percentage of CD16- and CD206-positive cells to CD68-positive cells in the outer annulus fibrosus layer. The number of vessels and nerve fibers and the severity of chronic inflammation with a focus on inflammatory cell infiltration, fibrosis, and capillary proliferation were also assessed., Results: The number of CD16-positive cells was the highest in type 2 IVDs, and was suppressed following the infiltration of CD206-positive cells. The degree of chronic inflammation was significantly higher in type 2 and type 3 IVDs, and the number of nerve fibers was significantly higher in type 3 IVDs. The endothelial cells of small vessels were positive for nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 expression. Staining for tropomyosin receptor kinase (Trk)-A, Trk-B, and Trk-C was positive in aberrant fibers. In immunoblot analysis, the expression levels of these neurotrophic factors and receptors were significantly higher in type 2 and 3 IVDs., Conclusions: The polarity of macrophages around newly developed microvasculature might be altered with cervical IVD degeneration. A higher number of infiltrating M1 macrophages around the vessels was associated with chronic inflammation; however, their number got suppressed following the infiltration of M2 macrophages. The expression of neurotrophins in the capillaries of small vessels might contribute to neural ingrowth into degenerated IVDs., Clinical Significance: Clarifying macrophages polarity change around new microvasculature associated with progression of IVD degeneration could enhance our understanding of the underlying mechanisms of neural ingrowth into degenerated IVDs and lead to development of a novel therapeutic target for prevention of IVD., Competing Interests: Declarations of competing interests All authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Previous research has identified an association between external radiation and disc degeneration, but the mechanism was poorly understood. This study explores the effects of ionizing radiation (IR) on inducing cellular senescence of annulus fibrosus (AF) in cell culture and in an in vivo mouse model. Exposure of AF cell culture to 10-15 Gy IR for 5 min followed by 5 days of culture incubation resulted in almost complete senescence induction as evidenced by SA-βgal positive staining of cells and elevated mRNA expression of the p16 and p21 senescent markers. IR-induced senescent AF cells exhibited increased matrix catabolism, including elevated matrix metalloproteinase (MMP)-1 and -3 protein expression and aggrecanolysis. Analogous results were seen with whole body IR-exposed mice, demonstrating that genotoxic stress also drives disc cellular senescence and matrix catabolism in vivo. These results have important clinical implications in the potential adverse effects of ionizing radiation on spinal health.

Intervertebral disc (IVD) degeneration is the main source of intractable lower back pain, and symptomatic IVD degeneration could be due to different degeneration mechanisms. In this article, we describe the molecular basis of symptomatic IVD degenerative disc diseases (DDDs), emphasizing the role of degeneration, inflammation, angiogenesis, and extracellular matrix (ECM) regulation during this process. In symptomatic DDD, pro-inflammatory mediators modulate catabolic reactions, resulting in changes in ECM homeostasis and, finally, neural/vascular ingrowth-related chronic intractable discogenic pain. In ECM homeostasis, anabolic protein-regulating genes show reduced expression and changes in ECM production, while matrix metalloproteinase gene expression increases and results in aggressive ECM degradation. The resultant loss of normal IVD viscoelasticity and a concomitant change in ECM composition are key mechanisms in DDDs. During inflammation, a macrophage-related cascade is represented by the secretion of high levels of pro-inflammatory cytokines, which induce inflammation. Aberrant angiogenesis is considered a key initiative pathologic step in symptomatic DDD. In reflection of angiogenesis, vascular endothelial growth factor expression is regulated by hypoxia-inducible factor-1 in the hypoxic conditions of IVDs. Furthermore, IVD cells undergoing degeneration potentially enhance neovascularization by secreting large amounts of angiogenic cytokines, which penetrate the IVD from the outer annulus fibrosus, extending deep into the outer part of the nucleus pulposus. Based on current knowledge, a multi-disciplinary approach is needed in all aspects of spinal research, starting from basic research to clinical applications, as this will provide information regarding treatments for DDDs and discogenic pain., Competing Interests: The authors have no potential conflicts of interest to disclose., (© Copyright: Yonsei University College of Medicine 2022.)

Oxidative stress and apoptosis play important roles in the pathogenesis of various degenerative diseases. Previous studies have shown that naringin can exert therapeutic effects in multiple degenerative diseases by resisting oxidative stress and inhibiting apoptosis. Although naringin is effective in treating degenerative disc disease, the underlying mechanism remains unclear. This study is aimed at investigating the effects of naringin on oxidative stress, apoptosis, and intervertebral disc degeneration (IVDD) induced by cyclic stretch and the underlying mechanisms in vitro and in vivo . Abnormal cyclic stretch was applied to rat annulus fibrosus cells, which were then treated with naringin, to observe the effects of naringin on apoptosis, oxidative stress, mitochondrial function, and the nuclear factor- (NF-) κ B signaling pathway. Subsequently, a rat model of IVDD induced by dynamic and static imbalance was established to evaluate the effects of naringin on the degree of degeneration (using imaging and histology), apoptosis, and oxidative stress in the serum and the intervertebral disc. Naringin inhibited the cyclic stretch-induced apoptosis of annulus fibrosus cells, reduced oxidative stress, improved mitochondrial function, enhanced the antioxidant capacity, and suppressed the activation of the NF- κ B signaling pathway. Additionally, it reduced the degree of IVDD (evaluated using magnetic resonance imaging) and the level of oxidative stress and inhibited apoptosis and p-P65 expression in the intervertebral discs of rats. Thus, naringin can inhibit cyclic stretch-induced apoptosis and delay IVDD, and the underlying mechanism may be related to the inhibition of oxidative stress and activation of the NF- κ B signaling pathway. Naringin may be an effective drug for treating degenerative disc disease., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2022 Yue-Hui Zhang et al.)

Intervertebral disc degeneration (IDD) is a leading contributor to low back pain. The intervertebral disc (IVD) is composed of three tissue types: the central gelatinous nucleus pulposus (NP) tissue, the surrounding annulus fibrosus (AF) tissue, and the inferior and superior cartilage endplates. The IVD microenvironment is hypoxic, acidic, hyperosmotic, and low in nutrients because it is mostly avascular. The cellular processes that underlie IDD initiation and progression are still poorly understood. Specifically, a lack of understanding regarding NP cell metabolism and physiology hinders the development of effective therapeutics to treat IDD patients. Autophagy is a vital intracellular degradation process that removes damaged organelles, misfolded proteins, and intracellular pathogens and recycles the degraded components for cellular energy and function. NP cells have adapted to survive within their harsh tissue microenvironment using processes that are largely unknown, and we postulate autophagy is one of these undiscovered mechanisms. In this review, we describe unique features of the IVD tissue, review how physiological stressors impact autophagy in NP cells in vitro, survey the current understanding of autophagy regulation in the IVD, and assess the relationship between autophagy and IDD. Published studies confirm autophagy markers are present in IVD tissue, and IVD cells can regulate autophagy in response to cellular stressors in vitro. However, data are still lacking to determine the exact mechanisms regulating autophagy in IVD cells. More in-depth research is needed to establish whether autophagy is necessary to maintain IVD cell health and validate autophagy as a relevant therapeutic target for treating IDD., (© 2021 Wiley Periodicals LLC.)

Back and neck pain are significant healthcare burdens that are commonly associated with pathologies of the intervertebral disc (IVD). The poor understanding of the cellular heterogeneity within the IVD makes it difficult to develop regenerative IVD therapies. To address this gap, we developed an atlas of bovine (Bos taurus) caudal IVDs using single-cell RNA-sequencing (scRNA-seq). Unsupervised clustering resolved 15 unique clusters, which we grouped into the following annotated partitions: nucleus pulposus (NP), outer annulus fibrosus (oAF), inner AF (iAF), notochord, muscle, endothelial, and immune cells. Analyzing the pooled gene expression profiles of the NP, oAF, and iAF partitions allowed us to identify novel markers for NP (CP, S100B, H2AC18, SNORC, CRELD2, PDIA4, DNAJC3, CHCHD7, and RCN2), oAF (IGFBP6, CTSK, LGALS1, and CCN3), and iAF (MGP, COMP, SPP1, GSN, SOD2, DCN, FN1, TIMP3, WDR73, and GAL) cells. Network analysis on subpopulations of NP and oAF cells determined that clusters NP1, NP2, NP4, and oAF1 displayed gene expression profiles consistent with cell survival, suggesting these clusters may uniquely support viability under the physiological stresses of the IVD. Clusters NP3, NP5, oAF2, and oAF3 expressed various extracellular matrix (ECM)-associated genes, suggesting their role in maintaining IVD structure. Lastly, transcriptional entropy and pseudotime analyses found that clusters NP3 and NP1 had the most stem-like gene expression signatures of the NP partition, implying these clusters may contain IVD progenitor cells. Overall, results highlight cell type diversity within the IVD, and these novel cell phenotypes may enhance our understanding of IVD development, homeostasis, degeneration, and regeneration., (© 2021 Federation of American Societies for Experimental Biology.)

Intervertebral disc degeneration is highly prevalent within the elderly population and is a leading cause of chronic back pain and disability. Due to the link between disc degeneration and senescence, we explored the ability of the Dasatinib and Quercetin drug combination (D + Q) to prevent an age-dependent progression of disc degeneration in mice. We treated C57BL/6 mice beginning at 6, 14, and 18 months of age, and analyzed them at 23 months of age. Interestingly, 6- and 14-month D + Q cohorts show lower incidences of degeneration, and the treatment results in a significant decrease in senescence markers p16 INK4a , p19 ARF , and SASP molecules IL-6 and MMP13. Treatment also preserves cell viability, phenotype, and matrix content. Although transcriptomic analysis shows disc compartment-specific effects of the treatment, cell death and cytokine response pathways are commonly modulated across tissue types. Results suggest that senolytics may provide an attractive strategy to mitigating age-dependent disc degeneration., (© 2021. The Author(s).)

Given the importance of the cartilage endplate (CEP) in low back pain (LBP), there is a need to characterize the human CEP at the molecular, cell, and tissue levels to inform treatment strategies that target it. The goal of this study was to characterize the structure, matrix composition, and cell phenotype of the human CEP compared with adjacent tissues within the intervertebral joint: the nucleus pulposus (NP), annulus fibrosus (AF), and articular cartilage (AC). Isolated CEP, NP, AF, and AC tissues and cells were evaluated for cell morphology, matrix composition, collagen structure, glycosaminoglycan content, and gene and protein expression. The CEP contained elongated cells that mainly produce a collagen-rich interterritorial matrix and a proteoglycan-rich territorial matrix. The CEP contained significantly fewer glycosaminoglycans than the NP tissue. Significant differences in matrix and cell marker gene expression were observed between CEP and NP or AF, with the greatest differences between CEP and AC. We were able to distinguish NP from CEP cells using collagen-10 (COLX), highlighting COLX as a potential CEP marker. Our findings suggest that at the cell and tissue levels, the CEP demonstrates both similarities and differences when compared with NP, AF, and hyaline AC. This study highlights a unique structure, matrix composition, and cell phenotype for the human CEP and can help to inform regenerative strategies that target the intervertebral disc joint in chronic LBP., (© 2020 Orthopaedic Research Society.)

It has been suggested that curvature progression in adolescent idiopathic scoliosis occurs through irreversible changes in the intervertebral discs. Strains of mice have been identified who differ in their disc wedging response upon extended asymmetrical compression. Annulus fibrosus (AF) tissue remodeling could contribute to the faster disc wedging progression previously observed in these mice. Differences in collagen remodeling capacity of AF cells between these in-bred mice strains were compared using an in vitro microtissue system. AF cells of 8-10-week-old LG/J ("fast-healing") and C57BL/6J ("normal healing") mice were embedded in a microtissue platform and cultured for 48 h. Hereafter, tissues were partially released and cultured for another 96 h. Microtissue surface area and waistcoat contraction, collagen orientation, and collagen content were measured. After 96 h postrelease, microtissues with AF cells of LG/J mice showed more surface area contraction (p < .001) and waistcoat contraction (p = .002) than C57BL/6J microtissues. Collagen orientation did not differ at 24 h after partial release. However, at 96 h, collagen in the microtissues from LG/J AF cells was aligned more than in those from C57BL/6J mice (p < .001). Collagen content did not differ between microtissues at 96 h. AF cells of inbred LG/J mice were better able to remodel and realign their collagen fibers than those from C57BL/6J mice. The remodeling of AF tissue could be contributing to the faster disc wedging progression observed in LG/J mice., (© 2020 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Yes-associated protein (YAP) activity responded to physical and mechanical cues such as extracellular matrix (ECM), cell density and the mechanical regulation of YAP controlled cellular proliferation and inhibition of apoptotic signals. The intervertebral disc (IVD) comprises a heterogeneous population of cells, including those of the nucleus pulposus (NP) and annulus fibrosus (AF), which are diverse in phenotype, partly due to the different ECM and mechanical loads they experience. How do IVD cells sense microenvironment and what is the relationship between YAP and cytoskeleton in the process of intervertebral disc degeneration (IDD) are not well understood. First, Hippo pathway and cytoskeleton organisation were assessed in the NP and AF of immature (4 weeks), mature (14 weeks), aged (50 weeks), and degenerated (14 weeks, 4 weeks after annulus puncture) IVDs. Second, to assess the effect of ECM composition and cell density on cytoskeleton and YAP levels, we seeded cells at different densities on three types of ECM. In this study, YAP and F-actin activity decreased gradually with age in natural IDD. Hippo signalling was suppressed in the early stages of disc injury, demonstrating the potential for endogenous repair, but this repair did not prevent further disc degeneration. β-tubulin and vimentin filaments provide the cell with its shape and its elastic properties in resisting mechanical forces. The Hippo pathway and cytoskeleton were shown to be regulated by cell density and the ECM composition., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Inflammation response is an important reason for disc cell senescence during disc degeneration. Recently, melatonin is suggested to protect against disc degeneration. However, the effects of melatonin on annulus fibrosus (AF) cell senescence are not fully studied. The main purpose of this study was to investigate the effects of melatonin on AF cell senescence in an inflammatory environment and the underlying mechanism. Rat disc AF cells were cultured in a medium with tumor necrosis factor- α (TNF- α ). Melatonin was added along with the medium to observe its protective effects. Compared with the control AF cells, TNF- α significantly declined cell proliferation potency and telomerase activity, elevated senescence-associated β -galactosidase (SA- β -Gal) activity, upregulated protein expression of senescence markers (p16 and p53), and increased reactive oxygen species (ROS) content and activity of the NF- κ B pathway. However, when the TNF- α -treated AF cells were incubated with melatonin, ROS content and activity of the NF- κ B pathway were decreased, and those parameters reflecting cell senescence indicated that AF cell senescence was also partly alleviated. Together, melatonin suppresses AF cell senescence through regulating the ROS/NF- κ B pathway in an inflammatory environment. This study sheds a new light that melatonin may be promising to retard inflammation-caused disc degeneration., Competing Interests: There are no conflicts of interest in this work., (Copyright © 2021 Jing Li et al.)

Mesenchymal stem/stromal cell (MSC)-based therapies have been proposed for back pain and disc degeneration, despite limited knowledge on their mechanism of action. The impact of MSCs/their secretome on annulus fibrosus (AF) cells and tissue was analysed in bovine AF organ cultures (AF-OCs) exposed to upper-physiological cyclic tensile strain (CTS, 9 %, 1 Hz, 3 h/d) and interleukin (IL)-1β in a custom-made device. A 4 d treatment of the CTS + IL-1β-stimulated AF-OCs with MSC secretome downregulated the expression of inflammation markers [IL-6, IL-8, prostaglandin-endoperoxide synthase 2 (PTGS2)], complement system regulators [cluster of differentiation (CD)46, CD55, CD59] and matrix metalloproteinase 1 but also of tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) and collagen type I. At the protein level, it was confirmed that IL-6, MMP-3 and collagen content was decreased in AF-OCs treated with the MSC secretome compared to the CTS + IL-1β stimulation alone. 9 d after treatment, a biomechanical peel-force test showed that the annular adhesive strength was significantly decreased by the MSC secretome treatment. Overall, MSC secretome had a stronger impact on AF tissue than MSCs in co-culture. The secretome contributed to a decrease in the inflammatory and catabolic status of AF cells activated by CTS + IL-1β and played a role in the regulation of the complement system. However, it also contributed to a decrease in collagen at the gene/protein level and in AF mechanical strength compared to the CTS + IL-1β stimulation alone. Therefore, the use of MSC secretome requires further investigation regarding its influence on disc matrix properties.

Background: Collagen cross-links contribute to the mechanical resilience of the intervertebral disc (IVD). UVA-light-activated riboflavin-induced collagen crosslinking (UVA-CXL) is a well-established and effective ophthalmological intervention that increases the mechanical rigidity of the collagen-rich corneal matrix in Keratoconus. This study explores the feasibility, safety and efficacy of translating this intervention in reinforcing the IVD., Methods: Annulus fibrosus (AF) cells were isolated from bovine IVDs and treated with different combinations of riboflavin (RF) concentrations (0.05-8 mM) and UVA light intensities (0.3-4 mW/cm2). Metabolic activity (resazurin assay), cell viability (TUNEL assay), and gene expression of apoptosis regulators C-FOS and PT5 were assessed immediately and 24 hours after treatment. Biomechanical effects of UVA-CXL on IVDs were measured by indentation analysis of changes in the instantaneous modulus and by peel-force delamination strength analysis of the AF prior and after treatment., Results: Different intensities of UVA did not impair the metabolic activity of AF cells. However, RF affected metabolic activity (p < 0.001). PT53 expression was similar in all RF conditions tested while C-FOS expression decreased 24 hours after treatment. Twenty-four hours after treatment, no apoptotic cells were observed in any condition tested. Biomechanical characterizations showed a significant increase in the annular peel strength of the UVA-CXL group, when compared to controls of UVA and RF alone (p < 0.05). UVA-CXL treated IVDs showed up to 152% higher (p < 0.001) instantaneous modulus values compared to the untreated control., Conclusion: This is the first study on UVA-CXL treatment of IVD. It induced significantly increased delamination strength and instantaneous modulus indentation values in intact IVD samples in a structure-function relationship. RF concentrations and UVA intensities utilized in ophthalmological clinical protocols were well tolerated by the AF cells. Our findings suggest that UVA-CXL may be a promising tool to reinforce the IVD matrix., Competing Interests: The authors have declared that no competing interests exist.

The intervertebral disc is the largest avascular low-nutrient organ in the body. Thus, resident cells may utilize autophagy, a stress-response survival mechanism, by self-digesting and recycling damaged components. Our objective was to elucidate the involvement of autophagy in rat experimental disc degeneration. In vitro, the comparison between human and rat disc nucleus pulposus (NP) and annulus fibrosus (AF) cells found increased autophagic flux under serum deprivation rather in humans than in rats and in NP cells than in AF cells of rats ( n = 6). In vivo, time-course Western blotting showed more distinct basal autophagy in rat tail disc NP tissues than in AF tissues; however, both decreased under sustained static compression ( n = 24). Then, immunohistochemistry displayed abundant autophagy-related protein expression in large vacuolated disc NP notochordal cells of sham rats. Under temporary static compression ( n = 18), multi-color immunofluorescence further identified rapidly decreased brachyury-positive notochordal cells with robust expression of autophagic microtubule-associated protein 1 light chain 3 (LC3) and transiently increased brachyury-negative non-notochordal cells with weaker LC3 expression. Notably, terminal deoxynucleotidyl transferase dUTP nick end labeling-positive apoptotic death was predominant in brachyury-negative non-notochordal cells. Based on the observed notochordal cell autophagy impairment and non-notochordal cell apoptosis induction under unphysiological mechanical loading, further investigation is warranted to clarify possible autophagy-induced protection against notochordal cell disappearance, the earliest sign of disc degeneration, through limiting apoptosis.

Embryonic muscle forces are necessary for normal vertebral development and spinal curvature, but their involvement in intervertebral disc (IVD) development remains unclear. The aim of the current study was to determine how muscle contractions affect (1) notochord involution and vertebral segmentation, and (2) IVD development including the mechanical properties and morphology, as well as collagen fibre alignment in the annulus fibrosus. Muscular dysgenesis (mdg) mice were harvested at three prenatal stages: at Theiler Stage (TS)22 when notochord involution starts, at TS24 when involution is complete, and at TS27 when the IVD is formed. Vertebral and IVD development were characterised using histology, immunofluorescence, and indentation testing. The results revealed that notochord involution and vertebral segmentation occurred independently of muscle contractions between TS22 and TS24. However, in the absence of muscle contractions, we found vertebral fusion in the cervical region at TS27, along with (i) a displacement of the nucleus pulposus towards the dorsal side, (ii) a disruption of the structural arrangement of collagen in the annulus fibrosus, and (iii) an increase in viscous behaviour of the annulus fibrosus. These findings emphasise the important role of mechanical forces during IVD development, and demonstrate a critical role of muscle loading during development to enable proper annulus fibrosus formation. They further suggest a need for mechanical loading in the creation of fibre-reinforced tissue engineering replacement IVDs as a therapy for IVD degeneration.

Background: Intervertebral disc degeneration contributes to low back pain. The avascular intervertebral disc consists of a central hypoxic nucleus pulpous (NP) surrounded by the more oxygenated annulus fibrosus (AF). Lactic acid, an abundant end-product of NP glycolysis, has long been viewed as a harmful waste that acidifies disc tissue and decreases cell viability and function. As lactic acid is readily converted into lactate in disc tissue, the objective of this study was to determine whether lactate could be used by AF cells as a carbon source rather than being removed from disc tissue as a waste byproduct., Methods: Import and conversion of lactate to tricarboxylic acid (TCA) cycle intermediates and amino acids in rabbit AF cells were measured by heavy-isotope ( 13 C-lactate) tracing experiments using mass spectrometry. Levels of protein expression of lactate converting enzymes, lactate importer and exporter in NP and AF tissues were quantified by Western blots. Effects of lactate on proteoglycan ( 35 S-sulfate) and collagen ( 3 H-proline) matrix protein synthesis and oxidative phosphorylation (Seahorse XFe96 Extracellular Flux Analyzer) in AF cells were assessed., Results: Heavy-isotope tracing experiments revealed that AF cells imported and converted lactate into TCA cycle intermediates and amino acids using in vitro cell culture and in vivo models. Addition of exogenous lactate (4 mM) in culture media induced expression of the lactate importer MCT1 and increased oxygen consumption rate by 50%, mitochondrial ATP-linked respiration by 30%, and collagen synthesis by 50% in AF cell cultures grown under physiologic oxygen (2-5% O 2 ) and glucose concentration (1-5 mM). AF tissue highly expresses MCT1, LDH-H, an enzyme that preferentially converts lactate to pyruvate, and PDH, an enzyme that converts pyruvate to acetyl-coA. In contrast, NP tissue highly expresses MCT4, a lactate exporter, and LDH-M, an enzyme that preferentially converts pyruvate to lactate., Conclusions: These findings support disc lactate-dependent metabolic symbiosis in which lactate produced by the hypoxic, glycolytic NP cells is utilized by the more oxygenated AF cells via oxidative phosphorylation for energy and matrix production, thus shifting the current research paradigm of viewing disc lactate as a waste product to considering it as an important biofuel. These scientifically impactful results suggest novel therapeutic targets in disc metabolism and degeneration.

The intervertebral disc (IVD) is a complex joint structure comprising three primary components-namely, nucleus pulposus (NP), annulus fibrosus (AF), and cartilaginous endplate (CEP). The IVD retrieves oxygen from the surrounding vertebral body through CEP by diffusion and likely generates ATP via anaerobic glycolysis. IVD degeneration is characterized by a cascade of cellular, compositional, structural changes. With advanced age, pronounced changes occur in the composition of the disc extracellular matrix (ECM). NP and AF cells in the IVD possess poor regenerative capacity compared with that of other tissues. Hypoxia-inducible factor (HIF) is a master transcription factor that initiates a coordinated cellular cascade in response to a low oxygen tension environment, including the regulation of numerous enzymes in response to hypoxia. HIF-1α is essential for NP development and homeostasis and is involved in various processes of IVD degeneration process, promotes ECM in NP, maintains the metabolic activities of NP, and regulates dystrophic mineralization of NP, as well as angiogenesis, autophagy, and apoptosis during IVD degeneration. HIF-1α may, therefore, represent a diagnostic tool for early IVD degeneration and a therapeutic target for inhibiting IVD degeneration.

Mechanical loading can induce or antagonize the extracellular matrix (ECM) synthesis, proliferation, migration, and inflammatory responses of annulus fibrosus cells (AFCs), depending on the loading mode and level. Caveolin-1 (Cav1), the core protein of caveolae, plays an important role in cellular mechanotransduction and inflammatory responses. In the present study, we presented that AFCs demonstrated different behaviors when subjected to cyclic tensile strain (CTS) for 24 h at a magnitude of 0%, 2%, 5% and 12%, respectively. It was found that 5% CTS had positive effects on cell proliferation, migration and anabolism, while 12% CTS had the opposite effects. Besides, cells exposed to interleukin-1β stimulus exhibited an increase expression in inflammatory genes, and the expression of these genes decreased after exposure to moderate mechanical loading with 5% CTS. In addition, 5% CTS decreased the level of Cav1 and integrin β1 and exhibited anti-inflammatory effects. Moreover, the expression of integrin β1 and p-p65 increased in AFCs transfected with Cav1 plasmids. In vivo results revealed that moderate mechanical stimulation could recover the water content and morphology of the discs. In conclusion, moderate mechanical stimulation restrained Cav1-mediated signaling pathway and exhibited anti-inflammatory effects on AFCs. Together with in vivo results, this study expounds the underlying molecular mechanisms on the effect of moderate mechanical stimulation on intervertebral discs (IVDs) and may provide a new therapeutic strategy for the treatment of IVD degeneration., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Ferroptosis is a necrotic form of regulated cell death that was associated with lipid peroxidation and free iron-mediated Fenton reactions. It has been reported that iron deficiency had been implicated in the pathogenesis of intervertebral disc degeneration (IVDD) by activating apoptosis. However, the role of ferroptosis in the process of IVDD has not been illuminated. Here, we demonstrate the involvement of ferroptosis in IVDD pathogenesis. Our in vitro models show the changes in protein levels of ferroptosis marker and enhanced lipid peroxidation level during oxidative stress. Safranin O staining, hematoxylin-eosin staining, and immunohistochemical were used to assess the IVDD after 8 weeks of surgical procedure in vivo. Treatment with ferrostatin-1, deferoxamine, and RSL3 demonstrate the role of ferroptosis in tert-butyl hydroperoxide (TBHP)-treated annulus fibrosus cells (AFCs) and nucleus pulposus cells (NPCs). Ferritinophagy, nuclear receptor coactivator 4 (NCOA4)-mediated ferritin selective autophagy, is originated during the process of ferroptosis in response to TBHP treatment. Knockdown and overexpression NCOA4 further prove TBHP may induce ferroptosis of AFCs and NPCs in an autophagy-dependent way. These findings support a role for oxidative stress-induced ferroptosis in the pathogenesis of IVDD., (© 2020 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Objective: Rat intervertebral disc (IVD) is one of the most commonly used and cost-effective alternative models for human IVD. Many IVD related clinical studies need to be pre-tested on rat IVDs. However, studies on the heterogeneous cell clusters of the rat IVD are inadequate, and a further understanding of the marker genes and cell phenotypes of healthy mature IVD cells is essential., Methods: In this study, we used the 10X Genomics technology to analyze the single-cell transcriptome of purified wild-type rat IVDs., Results: We identified potentially new gene markers of IVDs via single-cell sequencing. Based on the unsupervised cluster analysis of 13,578 single-cell transcripts, 3 known IVD cell types were identified. We provided a complete single-cell gene expression map of the IVD. Immunohistochemical and immunofluorescence images of rat disc sections confirmed the new marker genes of all cell types. One group of heterologous cell groups expressed multi-functional stem cell (MSC)-specific genes, indicating the stem cell potential of IVD cells., Conclusion: We provided the phenotype and marker genes of IVD cells at the single-cell level, reconfirmed existing data, and proposed new marker genes, including MSC marker genes. By identifying more accurate target cells and genes, our results pave the way for further study of the response of individual disc cells to disease states and provide the basis for future disc regeneration therapies., (Copyright © 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Background: This study aimed to develop a sensitive, accurate method for simultaneously quantifying cefuroxime and clindamycin in human serum, lumbar anulus fibrosus and nucleus pulposus., Methods: Cefuroxime and clindamycin were quantified using ultra high-performance liquid chromatography-electrospray ionization tandem mass spectrometry in multiple-reaction-monitoring mode on a triple-quadrupole AB Qtrap 5500 system in positive ion mode. Internal standards were D 3 -cefuroxime and D 3, 13 C-clindamycin. Samples were pretreated by precipitating total protein., Results: The method showed high sensitivity and good linearity over broad calibration ranges from 100 to 100 000 ng/mL for cefuroxime and 10 to 10 000 ng/mL for clindamycin in serum, and from 10 to 10 000 ng/mL for cefuroxime and 1 to 1 000 ng/mL for clindamycin in lumbar nucleus pulposus. In all sample types, correlation coefficients were greater than 0.99, intra- and inter-day precision (relative standard deviation) was less than 15%, and accuracy (relative error) was within 14% for both analytes. This method was effective at quantifying penetration of cefuroxime and clindamycin in patients undergoing oblique lumbar interbody fusion surgery., Conclusions: A very sensitive, specific method for simultaneous detection of cefuroxime and clindamycin has been developed for human lumbar anulus fibrosus, nucleus pulposus and serum samples., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aims: The intervertebral disc is the largest avascular organ of the body. Vascularization of the disc has been typically regarded as a pathological feature of intervertebral disc degeneration (IDD). However, the underlying mechanism of vascularization in IDD is still unclear. The current study aimed to investigate the role of AF cell derived exosome (AF-exo) in the interaction with human umbilical vein endothelial cells (HUVECs) and its potential role in the regulation of vascularization in IDD., Main Methods: Human AF tissues were obtained from patients with IDD and idiopathic scoliosis. The AF-exo were isolated and identified by transmission electron microscopy (TEM), nanoparticle trafficking analysis (NTA) and Western blotting. Then, the AF-exo were used for HUVECs cultures. The migration of HUVECs was observed in 2D and 3D cultures. The inflammatory phenotype of HUVECs was examined by Real-time PCR and enzyme-linked immunosorbent assay (ELISA). Additionally, apoptosis of HUVECs were analyzed by flow cytometry., Key Findings: Here, we for the first time found that AF cells could secrete AF-exo and that the AF-exo could be phagocytosed by HUVECs. Additionally, we found that degenerated AF-exo exerted pro-vascularization effect on HUVECs by promoting cell migration (in 2D and 3D cultures) and inflammatory factor expression including IL-6, TNF-α, MMP-3, MMP-13 and VEGF, whereas the application of non-degenerated AF-exo demonstrated inverse effects., Significance: These results showed that AF-exo is an essential regulator mediating intercellular communication between AF cells and HUVECs, suggesting its important role in vascularization in the intervertebral disc., (Copyright © 2020. Published by Elsevier Inc.)

Cadmium (Cd), a type of heavy metal that accumulates in the body because of smoking, mediates the toxic effect of smoking in many diseases, such as cardiovascular disease, osteoarthritis, and osteoporosis. However, the toxic effect of Cd on intervertebral disc tissues have not been reported. In the current study, we demonstrated that Cd induced the apoptosis of annulus fibrosus (AF) cells, which contributed to intervertebral disc degeneration (IVDD). Specifically, Cd induced the nuclear translocation of FoxO1a, which drives AF cells apoptosis through mitochondrial-related pathway. Phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signal pathway is also involved in this process. The combined use of LY29002, an inhibitor of PI3K, and small interfering RNA-targeting FoxO1a confirmed the relationship between the PI3K/AKT signal pathway and FoxO1a. In summary, present research explores the mechanism behind the contribution of smoking to IVDD and finds a new feasible target for preventing IVDD in smoking., (© 2020 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Background: Intervertebral disc degeneration (IDD) has high morbidity and high disability. Exosomes as a favorable candidate for IDD treatment is a hot spot of current research., Methods: Exosomes were obtained from bone marrow mesenchymal stem cells (BM-MSCs) of patients with non-open femoral fractures. Then, annulus fibrosus (AF) cells were treated with IL-1β, IL-1β combined with exosomes or IL-1β combined with exosomes and rapamycin. Flow cytometry and CCK-8 assay were performed to explore the apoptosis and cell proliferation. Quantitative real-time PCR and western blot were performed to detect the gene and protein expression., Results: Exosomes were obtained from BM-MSCs successfully. BM-MSC-derived exosomes suppressed IL-1β-induced inflammation and apoptosis and promoted cell proliferation of AF cells. BM-MSC-derived exosomes inhibited autophagy of AF cells by activating PI3K/AKT/mTOR signaling pathway. The effect of BM-MSC-derived exosomes on inflammation and apoptosis of AF cells was rescued by rapamycin., Conclusions: In conclusion, our study revealed that BM-MSC-derived exosomes inhibited IL-1β-induced inflammation and apoptosis of AF cells by suppressing autophagy., Competing Interests: Conflicts of Interest The authors declare no conflict of interest., (Copyright © 2020 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

To evaluate dominant cell-to-cell paracrine interactions, including those of human annulus fibrosus (AF), nucleus pulposus (NP), and endothelial cells (ECs), in the production of inflammatory mediators and catabolic enzymes, ECs was cultured in soluble factors derived from AF or NP cells (AFCM or NPCM, respectively) and vice versa. We analysed IL-6 and -8, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and -3, nerve growth factor (NGF)-β, and brain-derived neurotrophic factors (BDNFs) with qRT-PCR and ELISA. We implement a microfluidic platform to analyse migration properties of AF and NP cells and ECs in 3D cultures. Our results show that IL-1β-stimulated AF cells produced significantly higher levels of IL-6 and -8, VEGF, and MMP-1 than IL-1β-stimulated NP cells. However, production of IL-6 and -8, VEGF, and MMP-3 was significantly higher in NP cells than in AF cells, under the presence of ECs conditioned medium. We observed considerable migration of NP cells co-cultured with ECs through the microfluidic platform. These results suggest that AF cells may play a major role in the initial degeneration of intervertebral disc. Furthermore, it was found that interactions between NP cells and ECs may play a significant role in the development or progression of diseases.

Intervertebral disc (IVD) degeneration with chronic low back pain is associated with neo-vascularisation into the deeper IVD regions. During this process, endothelial cells (ECs), which are primarily responsible for angiogenesis, interact with the adjacent annulus fibrosus (AF) cells, which are the first line of defence against the invasion of vascular structures into deeper IVD regions. However, the accumulation of inflammatory and catabolic enzymes that results from this interaction promotes matrix degradation and an inflammatory response. Thus, regulating the production of these mediators and catabolic enzymes could ameliorate IVD degeneration. Photobiomodulation (PBM) therapy is a non-invasive stimulation known to have biologically beneficial effects on wound healing, tissue repair, and inflammation. Here, we examined the effects of PBM, administered at various wavelengths (645, 525, and 465 nm) and doses (16, 32, and 64 J/cm 2 ), on EC-stimulated human AF cells. Our results show that PBM selectively inhibited the EC-mediated production of inflammatory mediators, catabolic enzymes, and neurotrophins by human AF cells in a dose- and wavelength-dependent manner. These results suggest that PBM could be a superior and advanced treatment strategy for IVD degeneration.

Objective: Lack of specific marker-sets prohibits definition and functional distinction of cellular subtypes in the intervertebral disc (IVD), such as those from the annulus fibrosus (AF) and the nucleus pulposus (NP)., Design: We recently generated immortalized cell lines from human NP and AF tissues; these comprise a set of functionally distinct clonal subtypes. Whole transcriptome analyses were performed of 12 phenotypically distinct clonal cell lines (4× NP-Responder, 4× NP-nonResponder, 2× AF-Sheet forming, and 2× AF-nonSheet forming). Data sets were filtered for membrane-associated marker genes and compared to literature., Results: Comparison of our immortal cell lines to published primary NP, AF, and articular chondrocytes (AC) transcriptome datasets revealed preservation of AF and NP phenotypes. NP-specific membrane-associated genes were defined by comparison to AF cells in both the primary dataset (46 genes) and immortal cell-lines (161 genes). Definition of AF-specific membrane-associated genes yielded 125 primary AF cell and 92 immortal cell-line markers. Overlap between primary and immortal NP cells yielded high-confidence NP-specific marker genes for NP-R ( CLDN11, TMEFF2, CA12, ANXA2, CD44 ) and NP-nR (EFNA1, NETO2, SLC2A1). Overlap between AF and immortal AF subtypes yielded specific markers for AF-S ( COLEC12, LPAR1 ) and AF-nS ( CHIC1 )., Conclusions: The current study provides a reference platform for preclinical evaluation of novel membrane-associated cell type-specific markers in the IVD. Future research will focus on their biological relevance for IVD function in development, homeostasis, and degenerate conditions.

Intervertebral disc (IVD) herniation and degeneration is a major source of back pain. In order to regenerate a herniated and degenerated disc, closure of the anulus fibrosus (AF) is of crucial importance. For molecular characterization of AF, genome-wide Affymetrix HG-U133plus2.0 microarrays of native AF and cultured cells were investigated. To evaluate if cells derived from degenerated AF are able to initiate gene expression of a regenerative pattern of extracellular matrix (ECM) molecules, cultivated cells were stimulated with bone morphogenetic protein 2 (BMP2), transforming growth factor β1 (TGFβ1) or tumor necrosis factor-α (TNFα) for 24 h. Comparative microarray analysis of native AF tissues showed 788 genes with a significantly different gene expression with 213 genes more highly expressed in mild and 575 genes in severe degenerated AF tissue. Mild degenerated native AF tissues showed a higher gene expression of common cartilage ECM genes, whereas severe degenerated AF tissues expressed genes known from degenerative processes, including matrix metalloproteinases (MMP) and bone associated genes. During monolayer cultivation, only 164 differentially expressed genes were found. The cells dedifferentiated and altered their gene expression profile. RTD-PCR analyses of BMP2- and TGFβ1-stimulated cells from mild and severe degenerated AF tissue after 24 h showed an increased expression of cartilage associated genes. TNFα stimulation increased MMP1, 3, and 13 expression. Cells derived from mild and severe degenerated tissues could be stimulated to a comparable extent. These results give hope that regeneration of mildly but also strongly degenerated disc tissue is possible.

The intervertebral disc (IVD) degeneration is thought to be closely related to ingrowth of new blood vessels. However, the impact of anti-angiogenic factors in the maintenance of IVD avascularity remains unknown. Tenomodulin (Tnmd) is a tendon/ligament-specific marker and anti-angiogenic factor with abundant expression in the IVD. It is still unclear whether Tnmd contributes to the maintenance of IVD homeostasis, acting to inhibit vascular ingrowth into this normally avascular tissue. Herein, we investigated whether IVD degeneration could be induced spontaneously by the absence of Tnmd. Our results showed that Tnmd was expressed in an age-dependent manner primarily in the outer annulus fibrous (OAF) and it was downregulated at 6 months of age corresponding to the early IVD degeneration stage in mice. Tnmd knockout (Tnmd - / - ) mice exhibited more rapid progression of age-related IVD degeneration. These signs include smaller collagen fibril diameter, markedly lower compressive stiffness, reduced multiple IVD- and tendon/ligament-related gene expression, induced angiogenesis, and macrophage infiltration in OAF, as well as more hypertrophic-like chondrocytes in the nucleus pulposus. In addition, Tnmd and chondromodulin I (Chm1, the only homologous gene to Tnmd) double knockout (Tnmd - / - Chm1 - / - ) mice displayed not only accelerated IVD degeneration, but also ectopic bone formation of IVD. Lastly, the absence of Tnmd in OAF-derived cells promoted p65 and matrix metalloproteinases upregulation, and increased migratory capacity of human umbilical vein endothelial cells. In sum, our data provide clear evidences that Tnmd acts as an angiogenic inhibitor in the IVD homeostasis and protects against age-related IVD degeneration. Targeting Tnmd may represent a novel therapeutic strategy for attenuating age-related IVD degeneration., (© 2020 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Aim: To investigate the effects of metformin, a drug used widely for the treatment of type 2 diabetes mellitus, on human primary cell cultures prepared from uninjured segment of disc material intervertebral disk tissues., Material and Methods: Primary cell cultures were prepared using the tissues of six patients (three males and three females) who had undergone lumbar microdiscectomy and sequestrectomy. Untreated samples served as the control group, and metformintreated samples served as the experimental group. All the samples were evaluated using an inverted light microscope, acridine orange/propidium iodide staining (AO/PI), and a fluorescence microscope. The cytostatic and cytotoxic effects of metformin, which was administered to the samples using a commercial MTT assay kit, were also evaluated. The data obtained were statistically assessed, and the alpha significance value was accepted as less than 0.05. In addition, for the groups’ changes in the expressions of chondroadherin (CHAD), cartilage oligomeric matrix protein (COMP), interleukin-1β (IL-1β) matrix metalloproteinase 7 (MMP-7), and matrix metalloproteinase 19 (MMP-19), genes related to the extracellular matrix synthesis and degradation were determined using gene-specific TaqMan Gene Expression Assays., Results: The administration of the drug adversely affected nucleus pulposus (NP)/annulus fibrosus (AF) cells and extracellular matrixâ€"like structures. This was statistically significant (p < 0.05)., Conclusion: Clinicians should not disregard the adverse effects of metformin, which is used widely in clinical practice, on the components of intervertebral disk tissues.