Annulus Fibrosus Cells Research Articles

FUNCTIONAL ACTIVITY OF MONOMERIC C-REACTIVE PROTEIN IN INTERVERTEBRAL DISC

Monomeric C reactive protein (mCRP) presents important proinflammatory effects in endothelial cells, leukocytes, or chondrocytes. However, CRP in its pentameric form exhibits weak anti-inflammatory activity. It is used as a biomarker to follow severity and progression in infectious or inflammatory diseases, such as intervertebral disc degeneration (IVDD). This work assesses for the first time the mCRP effects in human intervertebral disc cells, trying to verify the pathophysiological relevance and mechanism of action of mCRP in the etiology and progression of IVD degeneration.We demonstrated that mCRP induces the expression of multiple proinflammatory and catabolic factors, like nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and lipocalin 2 (LCN2), in human annulus fibrosus (AF) and nucleus pulposus (NP) cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signaling of mCRP.Our results indicate that the effect of mCRP is persistent and sustained, regardless of the proinflammatory environment, as it was similar in healthy and degenerative human primary AF cells. This is the first article that demonstrates the localization of mCRP in intravertebral disc cells of the AF and NP and that provides evidence for the functional activity of mCRP in healthy and degenerative human AF and NP disc cells.

Rapamycin mitigates inflammation-mediated disc matrix homeostatic imbalance by inhibiting mTORC1 and inducing autophagy through Akt activation.

Low back pain is a global health problem that originated mainly from intervertebral disc degeneration (IDD). Autophagy, negatively regulated by the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, prevents metabolic and degenerative diseases by removing and recycling damaged cellular components. Despite growing evidence that autophagy occurs in the intervertebral disc, the regulation of disc cellular autophagy is still poorly understood. Annulus fibrosus (rAF) cell cultures derived from healthy female rabbit discs were used to test the effect of autophagy inhibition or activation on disc cell fate and matrix homeostasis. Specifically, different chemical inhibitors including rapamycin, 3-methyladenine, MK-2206, and PP242 were used to modulate activities of different proteins in the PI3K/Akt/mTOR signaling pathway to assess IL-1β-induced cellular senescence, apoptosis, and matrix homeostasis in rAF cells grown under nutrient-poor culture condition. Rapamycin, an inhibitor of mTOR complex 1 (mTORC1), reduced the phosphorylation of mTOR and its effector p70/S6K in rAF cell cultures. Rapamycin also induced autophagic flux as measured by increased expression of key autophagy markers, including LC3 puncta number, LC3-II expression, and cytoplasmic HMGB1 intensity and decreased p62/SQSTM1 expression. As expected, IL-1β stimulation promoted rAF cellular senescence, apoptosis, and matrix homeostatic imbalance with enhanced aggrecanolysis and MMP-3 and MMP-13 expression. Rapamycin treatment effectively mitigated IL-1β-mediated inflammatory stress changes, but these alleviating effects of rapamycin were abrogated by chemical inhibition of Akt and mTOR complex 2 (mTORC2). These findings suggest that rapamycin blunts adverse effects of inflammation on disc cells by inhibiting mTORC1 to induce autophagy through the PI3K/Akt/mTOR pathway that is dependent on Akt and mTORC2 activities. Hence, our findings identify autophagy, rapamycin, and PI3K/Akt/mTOR signaling as potential therapeutic targets for IDD treatment.

INVESTIGATION OF INTERVERTEBRAL DISC HERNIATION THROUGH A HUMAN ANULUS FIBROSUS PRO-INFLAMMATORY MODEL

Intervertebral disc (IVD) herniation involves a chronic inflammatory process and failure of the annulus fibrosus (AF), in a process still under investigated. Inflammation can lead to disorganization of extracellular matrix (ECM) and fibrotic alterations, among others. Most of the ongoing research relies on in vivo or ex vivo models that do not truly mimic the human microenvironment. Here, we developed a 3D in vitro model aimed at recapitulating inflammation progression in human AF (hAF) to improve the understanding of AF failure.hAF of herniated samples from low back pain patients were collected and digested as previously established. hAF cells were cultured in 2D and 3D, embedded in collagen-type 1 hydrogels. hAF cells in 2D and 3D were then stimulated with IL-1β (10ng/mL), in acute (48h) and chronic (7days) conditions. After 7 days, cell morphology together with extracellular matrix (ECM) (collagen I and fibronectin) were assessed by immunofluorescence. Inflammatory cytokine secretion were analyzed by a protein array.2D experiments showed alteration of cell morphology, with cell area reduction upon inflammatory stimulation. The profile of inflammatory cytokines secreted showed that: i) IL-1α increases with time in culture, while TNF-α decreases; and ii) IL-1α and MIG/CXCL9 are increased in chronic vs acute inflammation. ECM analysis showed no differences. In 3D culture, AF cell morphologic alterations with inflammation were also observed. Concerning ECM production, collagen I production was reduced with chronic inflammation, while fibronectin remained unaltered. Collagen II and aggrecan appear to increase with chronic inflammation. Cytokine production from 3D culture is currently being assessed. ECM alterations are being complemented by characterization at biomechanical (nanoindentation) level.Our data show that chronic inflammation is crucial to understand AF failure mechanisms and improve the resemblance of human IVD herniation.

Cadmium-induced annulus fibrosus cell senescence contributes to intervertebral disc degeneration via the JNK/p53 signaling pathway.

Investigating the impact of cadmium (Cd) on annulus fibrosus (AF) cells and its potential mechanism was the purpose of the current study. Cd was cultivated in different concentrations (0, 1, 5, 10, and 20 μM) on AF cells and the potential effects of the metal were assessed. Using the CCK-8 method, cell viability and proliferation were identified. Using transcriptome analysis, the annulus fibrosus cells were sequenced both with and without cadmium chloride. The EdU method was used to determine the rate of cell proliferation; senescence-associated β-galactosidase (SA-β-Gal) staining was used to determine the number of positive cells; and western blot, RT-PCR, and immunofluorescence were used to determine the protein and mRNA expression of senescence-associated proteins (p16, p21, and p53) and c-Jun N-terminal kinase (JNK). According to the findings, Cd has the ability to increase the production of senescence-associated genes (p16 and p21) and senescence-associated secreted phenotype (SASP), which includes IL-1β and IL-6. Through the JNK/p53 signal pathway, Cd exposure simultaneously accelerated AF cell senescence and promoted SASP. Following JNK inhibitor (SP600125) treatment, the expression of p53, JNK, and senescence-associated indices were all down-regulated. By activating the JNK/p53 signaling pathway, Cd can induce oxidative stress damage and AF cell senescence. These findings could provide a new approach for treating and preventing intervertebral disc degeneration (IVDD) caused by Cd exposure.

Enhanced Cell Viability and Migration of Primary Bovine Annular Fibrosus Fibroblast-like Cells Induced by Microsecond Pulsed Electric Field Exposure.

This study is the first to report the enhancement of cell migration and proliferation induced by in vitro microsecond pulsed electric field (μsPEF) exposure of primary bovine annulus fibrosus (AF) fibroblast-like cells. AF primary cells isolated from fresh bovine intervertebral disks (IVDs) are exposed to 10 and 100 μsPEFs with different numbers of pulses and applied electric field strengths. The results indicate that 10 μs-duration pulses induce reversible electroporation, while 100 μs pulses induce irreversible electroporation of the cells. Additionally, μsPEF exposure increased AF cell proliferation up to 150% while increasing the average migration speed by 0.08 μm/min over 24 h. The findings suggest that the effects of PEF exposure on cells are multifactorial-depending on the duration, intensity, and number of pulses used in the stimulation. This highlights the importance of optimizing the μsPEF parameters for specific cell types and applications. For instance, if the goal is to induce cell death for cancer treatment, then high numbers of pulses can be used to maximize the lethal effects. On the other hand, if the goal is to enhance cell proliferation, a combination of the number of pulses and the applied electric field strength can be tuned to achieve the desired outcome. The information gleaned from this study can be applied in the future to in vitro cell culture expansion and tissue regeneration.

Melatonin-loaded self-healing hydrogel targets mitochondrial energy metabolism and promotes annulus fibrosus regeneration

Intervertebral disc (IVD) herniation is a major cause of chronic low back pain and disability. The current nucleus pulposus (NP) discectomy effectively relieves pain symptoms, but the annulus fibrosus (AF) defects are left unrepaired. Tissue engineering approaches show promise in treating AF injury and IVD degeneration; however, the presence of an inflammatory milieu at the injury site hinders the mitochondrial energy metabolism of AF cells, resulting in a lack of AF regeneration. In this study, we fabricated a dynamic self-healing hydrogel loaded with melatonin (an endocrine hormone well-known for its antioxidant and anti-inflammatory properties) and investigate whether melatonin-loaded hydrogel could promote AF defect repair by rescuing the matrix synthesis and energy metabolism of AF cells. The protective effects of melatonin on matrix components (e.g. type I and II collagen and aggrecan) in AF cells were observed in the presence of interleukin (IL)-1β. Additionally, melatonin was found to activate the nuclear factor erythroid 2-related factor signaling pathway, thereby safeguarding the mitochondrial function of AF cells from IL-1β, as evidenced by the increased level of adenosine triphosphate, mitochondrial membrane potential, and respiratory chain factor expression. The incorporation of melatonin into a self-healing hydrogel based on thiolated gelatin and β-cyclodextrin was proposed as a means of promoting AF regeneration. The successful implantation of melatonin-loaded hydrogel has been shown to facilitate in situ regeneration of AF tissue, thereby impeding IVD degeneration by preserving the hydration of nucleus pulposus in a rat box-cut IVD defect model. These findings offer compelling evidence that the development of a melatonin-loaded dynamic self-healing hydrogel can promote the mitochondrial functions of AF cells and represents a promising strategy for IVD regeneration.

Inhibition of PP2A ameliorates intervertebral disc degeneration by reducing annulus fibrosus cells apoptosis via p38/MAPK signal pathway

BackgroundIntervertebral 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. MethodsAF 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 H2O2 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. ResultsThe expression of PP2A was increased in rat AF cells after H2O2 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. ConclusionsThis 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.

Advanced glycation end products promote intervertebral disc degeneration by transactivation of matrix metallopeptidase genes

Examine the mechanism by which advanced glycation end products (AGEs) induce intervertebral disc degeneration (IDD) in C57BL/6J mice. Matrix metallopeptidase (MMP) gene mRNA levels were assessed using RT-qPCR. Immunoprecipitation and co-immunoprecipitation were performed to identify the transcriptional complex regulating MMP expression due to AGEs. The preventive effects of inhibitors targeting this complex were tested in mice on high AGE diets. IDD and AGE accumulation were evident in mice on high-AGE diets (HAGEs), persisting across dietary shifts but absent in mice exclusively on low-AGE diets. Molecularly, HAGEs activated p21-activated kinase 1 (PAK1), prompting peroxisome proliferator-activated receptor gamma coactivator-related protein 1 (PPRC1) phosphorylation. Ubiquitin-specific protease 12 (USP12) interacted with the phosphorylated PPRC1 (pPPRC1), safeguarding it from proteasomal degradation. This pPPRC1, in collaboration with two histone acetyltransferases p300/CREB-binding protein (CBP) and a transcription factor activator protein 1(AP1), enhanced the expression of 12 MMP genes (MMP1a/1b/3/7/9/10/12/13/16/19/23/28). In vitro AGE exposure on nucleus pulposus and annulus fibrosus cells replicated this gene activation pattern, driven by the PAK1/pPPRC1-p300/CBP-AP1 pathway. The application of PAK1, p300, and AP1 inhibitors reduced pPPRC1-p300/CBP-AP1 binding to MMP promoters, diminishing their expression. These inhibitors effectively thwarted IDD in HAGE mice. Our results revealed that HAGEs instigate IDD via the PAK1/pPPRC1-p300/CBP-AP1 signaling pathway. This insight can guide therapeutic strategies to slow IDD progression in prediabetic/diabetic patients.

Sustained release of basic fibroblast growth factor in micro/nanofibrous scaffolds promotes annulus fibrosus regeneration.

Tissue engineering has promising applications in the treatment of intervertebral disc degeneration (IDD). The annulus fibrosus (AF) is critical for maintaining the physiological function of the intervertebral disc (IVD), but the lack of vessels and nutrition in AF makes it difficult to repair. In this study, we used hyaluronan (HA) micro-sol electrospinning and collagen type I (Col-I) self-assembly techniques to fabricate layered biomimetic micro/nanofibrous scaffolds, which released basic fibroblast growth factor (bFGF) to promote AF repair and regeneration after discectomy and endoscopic transforaminal discectomy. The bFGF enveloped in the core of the poly-L-lactic-acid (PLLA) core-shell structure was released in a sustained manner and promoted the adhesion and proliferation of AF cells (AFCs). Col-I could self-assemble on the shell of the PLLA core-shell scaffold to mimic the extracellular matrix (ECM) microenvironment, providing structural and biochemical cues for the regeneration of AF tissue. The in vivo studies showed that the micro/nanofibrous scaffolds promoted the repair of AF defects by simulating the microstructure of native AF tissue and inducing endogenous regeneration mechanism. Taken together, the biomimetic micro/nanofibrous scaffolds have clinical potential for the treatment of AF defects caused by IDD. STATEMENT OF SIGNIFICANCE: The annulus fibrosus (AF) is essential for the intervertebral disc(IVD) physiological function, yet it lacks vascularity and nutrition, making repair difficult. Micro-sol electrospinning technology and collagen type I (Col-I) self-assemblytechniquewere combined in this study to create a layered biomimetic micro/nanofibrous scaffold that releases basic fibroblast growth factor (bFGF) to promote AF repair and regeneration. Col-I could mimic the extracellular matrix (ECM) microenvironment,in vivo, offering structural and biochemical cues for AF tissue regeneration. This research indicates that micro/nanofibrous scaffolds have clinical potential for treating AF deficits induced by IDD.

The clock transcription factor BMAL1 is a key regulator of extracellular matrix homeostasis and cell fate in the intervertebral disc

The circadian clock in mammals temporally coordinates physiological and behavioural processes to anticipate daily rhythmic changes in their environment. Chronic disruption to circadian rhythms (e.g., through ageing or shift work) is thought to contribute to a multitude of diseases, including degeneration of the musculoskeletal system. The intervertebral disc (IVD) in the spine contains circadian clocks which control ∼6% of the transcriptome in a rhythmic manner, including key genes involved in extracellular matrix (ECM) homeostasis. However, it remains largely unknown to what extent the local IVD molecular clock is required to drive rhythmic gene transcription and IVD physiology. In this work, we identified profound age-related changes of ECM microarchitecture and an endochondral ossification-like phenotype in the annulus fibrosus (AF) region of the IVD in the Col2a1-Bmal1 knockout mice. Circadian time series RNA-Seq of the whole IVD in Bmal1 knockout revealed loss of circadian patterns in gene expression, with an unexpected emergence of 12 h ultradian rhythms, including FOXO transcription factors. Further RNA sequencing of the AF tissue identified region-specific changes in gene expression, evidencing a loss of AF phenotype markers and a dysregulation of ECM and FOXO pathways in Bmal1 knockout mice. Consistent with an up-regulation of FOXO1 mRNA and protein levels in Bmal1 knockout IVDs, inhibition of FOXO1 in AF cells suppressed their osteogenic differentiation. Collectively, these data highlight the importance of the local molecular clock mechanism in the maintenance of the cell fate and ECM homeostasis of the IVD. Further studies may identify potential new molecular targets for alleviating IVD degeneration.

Nonviral overexpression of Scleraxis or Mohawk drives reprogramming of degenerate human annulus fibrosus cells from a diseased to a healthy phenotype.

Intervertebral disc (IVD) degeneration is a major contributor to low back pain (LBP), yet there are no clinical therapies targeting the underlying pathology. The annulus fibrosus (AF) plays a critical role in maintaining IVD structure/function and undergoes degenerative changes such as matrix catabolism and inflammation. Thus, therapies targeting the AF are crucial to fully restore IVD function. Previously, we have shown nonviral delivery of transcription factors to push diseased nucleus pulposus cells to a healthy phenotype. As a next step in a proof-of-concept study, we report the use of Scleraxis (SCX) and Mohawk (MKX), which are critical for the development, maintenance, and regeneration of the AF and may have therapeutic potential to induce a healthy, pro-anabolic phenotype in diseased AF cells. MKX and SCX plasmids were delivered via electroporation into diseased human AF cells from autopsy specimens and patients undergoing surgery for LBP. Transfected cells were cultured over 14 days and assessed for cell morphology, viability, density, gene expression of key phenotypic, inflammatory, matrix, pain markers, and collagen accumulation. AF cells demonstrated a fibroblastic phenotype posttreatment. Moreover, transfection of SCX and MKX resulted in significant upregulation of the respective genes, as well as SOX9. Transfected autopsy cells demonstrated upregulation of core extracellular matrix markers; however, this was observed to a lesser effect in surgical cells. Matrix-degrading enzymes and inflammatory cytokines were downregulated, suggesting a push toward a pro-anabolic, anti-inflammatory phenotype. Similarly, pain markers were downregulated over time in autopsy cells. At the protein level, collagen content was increased in both MKX and SCX transfected cells compared to controls. This exploratory study demonstrates the potential of MKX or SCX to drive reprogramming in mild to moderately degenerate AF cells from autopsy and severely degenerate AF cells from surgical patients toward a healthy phenotype and may be a potential nonviral gene therapy for LBP.

Metabolomic signature and molecular profile of normal and degenerated human intervertebral disc cells

BACKGROUND CONTEXTIntervertebral 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. PURPOSECharacterize 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 DESIGNIntervertebral 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. METHODSThe 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. RESULTSLipidomic 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). CONCLUSIONSAltogether, 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 SIGNIFICANCEOur 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.

Moderate mechanical stimulation antagonizes inflammation of annulus fibrosus cells through YAP-mediated suppression of NF-κB signaling.

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.

Celecoxib alleviates nociceptor sensitization mediated by interleukin-1beta-primed annulus fibrosus cells

PurposeThis study aims to analyze the effect of pro-inflammatory cytokine-stimulated human annulus fibrosus cells (hAFCs) on the sensitization of dorsal root ganglion (DRG) cells. We further hypothesized that celecoxib (cxb) could inhibit hAFCs-induced DRG sensitization.MethodshAFCs from spinal trauma patients were stimulated with TNF-α or IL-1β. Cxb was added on day 2. On day 4, the expression of pro-inflammatory and neurotrophic genes was evaluated using RT-qPCR. Levels of prostaglandin E2 (PGE-2), IL-8, and IL-6 were measured in the conditioned medium (CM) using ELISA. hAFCs CM was then applied to stimulate the DRG cell line (ND7/23) for 6 days. Then, calcium imaging (Fluo4) was performed to evaluate DRG cell sensitization. Both spontaneous and bradykinin-stimulated (0.5 μM) calcium responses were analyzed. The effects on primary bovine DRG cell culture were performed in parallel to the DRG cell line model.ResultsIL-1ß stimulation significantly enhanced the release of PGE-2 in hAFCs CM, while this increase was completely suppressed by 10 µM cxb. hAFCs revealed elevated IL-6 and IL-8 release following TNF-α and IL-1β treatment, though cxb did not alter this. The effect of hAFCs CM on DRG cell sensitization was influenced by adding cxb to hAFCs; both the DRG cell line and primary bovine DRG nociceptors showed a lower sensitivity to bradykinin stimulation.ConclusionCxb can inhibit PGE-2 production in hAFCs in an IL-1β-induced pro-inflammatory in vitro environment. The cxb applied to the hAFCs also reduces the sensitization of DRG nociceptors that are stimulated by the hAFCs CM.

IN VITRO CATABOLIC PHENOTYPE INDUCTION OF INTERVERTEBRAL DISC CELLS IN 3D ALGINATE CULTURE

Previous research has shown catabolic cell signalling induced by TNF-α and IL-1β within intervertebral (IVD) cells. However, these studies have investigated this in 2D monolayer cultures, and under hyper-physiological doses. Thus, we aim to revisit the catabolic responses of bovine IVD cells in vitro in 3D culture under increasing doses of TNF-α or IL-1β stimulation at three different timepoints.Primary bovine nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated and expanded for two weeks. Subsequently, NP and AF cells were encapsulated in 1.2% alginate beads (4 × 106 cells/ml) and cultured for two weeks for phenotype recovery. Re-differentiated cells were stimulated with 0.1, 1 and 10 ng/ml TNF-α or with 0.01, 0.1 and 10 ng/ml IL-1β for one week. Beads were collected on the stimulation day (Day 0) and on Day 1 and 7 after stimulation.A dose-dependent upregulation of catabolic markers was observed in both cell types after one day of TNF-α or IL-1β stimulation. 10 ng/ml TNF-α stimulation induced a significant upregulation (p<0.05) of ADAMTS4, MMP3 and MMP13 in AF cells after one day of stimulation. Similarly, MMP3 upregulation showed a strong trend (p=0.0643) in NP cells. However, no effects on expression were seen after seven days. In addition, no significant difference between treatments in COL2, COL1 and ACAN expression was observed, and cell viability was not reduced at any time point, regardless of the treatment.We demonstrate a dose-dependent upregulation of catabolic markers in NP and AF cells under TNF-α or IL-1β stimulation, with a significant upregulation of ADAMTS4, MMP3 and MMP13 genes in AF cells after one day of treatment. Notably, after seven days of treatment, the dose-dependent effects were no longer observed possibly due to an adaptation mechanism of IVD cells to counter the metabolic shift.

Terminal Complement Activation Is Induced by Factors Released from Endplate Tissue of Disc Degeneration Patients and Stimulates Expression of Catabolic Enzymes in Annulus Fibrosus Cells.

Terminal complement complex (TCC) deposition was identified in human degenerated discs. To clarify the role of terminal complement activation in disc degeneration (DD), we investigated respective activating mechanisms and cellular effects in annulus fibrosus (AF) cells. Isolated cells from human AF, nucleus pulposus (NP), and endplate (EP) were stimulated with human serum alone or with zymosan and treated with either the C3 inhibitor Cp40 or the C5 antibody eculizumab. Complement activation was determined via anaphylatoxin generation and TCC deposition detection. Thereby, induced catabolic effects were evaluated in cultured AF cells. Moreover, C5 cleavage under degenerative conditions in the presence of AF cells was assessed. Zymosan-induced anaphylatoxin generation and TCC deposition was significantly suppressed by both complement inhibitors. Zymosan induced gene expression of ADAMTS4, MMP1, and COX2. Whereas the C3 blockade attenuated the expression of ADAMTS4, the C5 blockade reduced the expression of ADAMTS4, MMP1, and COX2. Direct C5 cleavage was significantly enhanced by EP conditioned medium from DD patients and CTSD. These results indicate that terminal complement activation might be functionally involved in the progression of DD. Moreover, we found evidence that soluble factors secreted by degenerated EP tissue can mediate direct C5 cleavage, thereby contributing to complement activation in degenerated discs.

Monomeric CRP regulates inflammatory responses in human intervertebral disc cells.

CRP is an acute-phase protein that is used as a biomarker to follow severity and progression in infectious and inflammatory diseases. Its pathophysiological mechanisms of action are still poorly defined. CRP in its pentameric form exhibits weak anti-inflammatory activity. The monomeric isoform (mCRP) exerts potent proinflammatory properties in chondrocytes, endothelial cells, and leucocytes. No data exist regarding mCRP effects in human intervertebral disc (IVD) cells. This work aimed to verify the pathophysiological relevance of mCRP in the aetiology and/or progression of IVD degeneration. We investigated the effects of mCRP and the signalling pathways that are involved in cultured human primary annulus fibrosus (AF) cells and in the human nucleus pulposus (NP) immortalized cell line HNPSV-1. We determined messenger RNA (mRNA) and protein levels of relevant factors involved in inflammatory responses, by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. We also studied the presence of mCRP in human AF and NP tissues by immunohistochemistry. We demonstrated that mCRP increases nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and Lipocalin 2 (LCN2) expression in human AF and NP cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signalling of mCRP. Finally, we demonstrated the presence of mCRP in human AF and NP tissues. Our results indicate, for the first time, that mCRP can be localized in IVD tissues, where it triggers a proinflammatory and catabolic state in degenerative and healthy IVD cells, and that NF-κβ signalling may be implicated in the mediation of this mCRP-induced state.

Electrical impedance decreases in annulus fibrosus cartilage exposed to microsecond pulsed electric fields ex vivo

Electropermeabilization of biomembranes often is measured by microscopic imaging of a membrane-impermeable fluorophore that penetrates the cells following pulsed electric field (PEF) exposure. PEF exposure subsequently changes physiological properties of tissue. One way to probe these changes in tissue is measuring electrical properties by way of electrochemical impedance spectroscopy (EIS). In this study, we analyse impedance and conductivity of bovine annulus fibrosus (AF) cartilage before and after exposures to PEF of 100 μs duration. Two PEF parameters–electric field amplitude and number of pulses–are varied, and total specific dose of PEF is calculated. AF tissue conductivity increases with both amplitude and number of pulses, indicating electropermeabilization of the AF cells. A Live/Dead cell imaging assay validates the EIS measurements, indicating intratissue cell permeabilization by μsPEF exposure. These results support the extension of EIS to monitor extent of electropermeabilization of cells within cartilage tissue.

Single-cell RNA sequencing reveals resident progenitor and vascularization-associated cell subpopulations in rat annulus fibrosus.

One of the main causes of low back pain is intervertebral disc degeneration (IDD). Annulus fibrosus (AF) is important for the integrity and functions of the intervertebral disc (IVD). However, the resident functional cell components such as progenitors and vascularization-associated cells in AF are yet to be fully identified. Identification of functional AF cell subpopulations including resident progenitors and vascularization-associated cells. In this study, the single-cell RNA sequencing data of rat IVDs from a public database were analyzed using Seurat for cell clustering, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for functional analysis, StemID for stem cell identification, Monocle and RNA velocity for pseudotime differentiation trajectory validation, single-cell regulatory network inference and clustering (SCENIC) for gene regulatory network (GRN) analysis, and CellChat for cell-cell interaction analysis. Immunostaining on normal and degenerated rat IVDs, as well as human AF, was used for validations. From the data analysis, seven AF cell clusters were identified, including two newly discovered functional clusters, the Grem1 + subpopulation and the Lum +​subpopulation. The Grem1 + subpopulation had progenitor characteristics, while the Lum +​subpopulation was associated with vascularization during IDD. The GRN analysis showed that Sox9 and Id1 were among the key regulators in the Grem1 + subpopulation, and Nr2f2 and Creb5 could be responsible for the vascularization function in the Lum +​subpopulation. Cell-cell interaction analysis revealed highly regulated cellular communications between these cells, and multiple signaling networks including PDGF and MIF signaling pathways were involved in the interactions. Our results revealed two new functional AF cell subpopulations, with stemness and vascularization induction potential, respectively. These findings complement our knowledge about IVDs, especially the AF, and in return provide potential cell source and regulation targets for IDD treatment and tissue repair. The existence of the cell subpopulations was also validated in human AF, which strengthen the clinical relevance of the findings.

Ccn2a-FGFR1-SHH signaling is necessary for intervertebral disc homeostasis and regeneration in adult zebrafish.

Intervertebral disc (IVD) degeneration is the primary cause of back pain in humans. However, the cellular and molecular pathogenesis of IVD degeneration is poorly understood. This study shows that zebrafish IVDs possess distinct and non-overlapping zones of cell proliferation and cell death. We find that, in zebrafish, cellular communication network factor 2a (ccn2a) is expressed in notochord and IVDs. Although IVD development appears normal in ccn2a mutants, the adult mutant IVDs exhibit decreased cell proliferation and increased cell death leading to IVD degeneration. Moreover, Ccn2a overexpression promotes regeneration through accelerating cell proliferation and suppressing cell death in wild-type aged IVDs. Mechanistically, Ccn2a maintains IVD homeostasis and promotes IVD regeneration by enhancing outer annulus fibrosus cell proliferation and suppressing nucleus pulposus cell death through augmenting FGFR1-SHH signaling. These findings reveal that Ccn2a plays a central role in IVD homeostasis and regeneration, which could be exploited for therapeutic intervention in degenerated human discs.