Human Intervertebral Disc Tissue Research Articles

Pip5k1γ promotes anabolism of nucleus pulposus cells and intervertebral disc homeostasis by activating CaMKII-Ampk pathway in aged mice.

Degenerative disc disease (DDD) represents a significant global health challenge, yet its underlying molecular mechanisms remain elusive. This study aimed to investigate the role of type 1 phosphatidylinositol 4-phosphate 5-kinase (Pip5k1) in intervertebral disc (IVD) homeostasis and disease. All three Pip5k1 isoforms, namely Pip5k1α, Pip5k1β, and Pip5k1γ, were detectable in mouse and human IVD tissues, with Pip5k1γ displaying a highest expression in nucleus pulposus (NP) cells. The expression of Pip5k1γ was significantly down-regulated in the NP cells of aged mice and patients with severe DDD. To determine whether Pip5k1γ expression is required for disc homeostasis, we generated a Pip5k1γfl/fl; AggrecanCreERT2 mouse model for the conditional knockout of the Pip5k1γ gene in aggrecan-expressing IVD cells. Our findings revealed that the conditional deletion of Pip5k1γ did not affect the disc structure or cellular composition in 5-month-old adult mice. However, in aged (15-month-old) mice, this deletion led to several severe degenerative disc defects, including decreased NP cellularity, spontaneous fibrosis and cleft formation, and a loss of the boundary between NP and annulus fibrosus. At the molecular level, the absence of Pip5k1γ reduced the anabolism of NP cells without markedly affecting their catabolic or anti-catabolic activities. Moreover, the loss of Pip5k1γ significantly dampened the activation of the protective Ampk pathway in NP cells, thereby accelerating NP cell senescence. Notably, Pip5k1γ deficiency blunted the effectiveness of metformin, a potent Ampk activator, in activating the Ampk pathway and mitigating lumbar spine instability (LSI)-induced disc lesions in mice. Overall, our study unveils a novel role for Pip5k1γ in promoting anabolism and maintaining disc homeostasis, suggesting it as a potential therapeutic target for DDD.

Sympathetic Neurotransmitter, VIP, Delays Intervertebral Disc Degeneration via FGF18/FGFR2-Mediated Activation of Akt Signaling Pathway.

Neuromodulation-related intervertebral disc degeneration (IVDD) is a novel IVDD pattern and are proposed recently. However, the mechanistic basis of neuromodulation and intervertebral disc (IVD) homeostasis remains unclear. Here, this study aimed to investigate the expression of postganglionic sympathetic nerve fiber-derived vasoactive intestinal peptide (VIP) system in human IVD tissue, and to assess the role of VIP-related neuromodulation in IVDD. Patient samples and in vitro cell experiments showed that the expression of receptors for VIP is negatively correlated with the severity of IVDD, and the administration of exogenous VIP can ameliorate interleukin 1β-induced nucleus pulposus (NP) cell apoptosis and inflammation. Further mRNA-seq analysis revealed that fibroblast growth factor 18- (FGF18)-mediated activation of V-akt murine thymoma viral oncogene homolog signaling pathway is involved in the protective effects of VIP on inflammation-induced NP cell degeneration. Further analysis identified VIP via its receptor vasoactive intestinal peptide receptor 2 can directly result in decreased expression of miR-15a-5p, which targeted FGF18. Finally, in vivo mice lumbar IVDD model confirmed that focally exogenous administration of VIP can effectively ameliorated the progression of IVDD, as shown by the radiological and histological analysis. In conclusion, these results indicated that sympathetic neurotransmitter, VIP, delayed IVDD via FGF18/FGFR2-mediated activation of V-akt murine thymoma viral oncogene homolog signaling pathway, which will broaden the horizon concerning how the neuromodulation correlates with IVDD and shed new light on novel therapeutical alternatives to IVDD.

Cynarin alleviates intervertebral disc degeneration via protecting nucleus pulposus cells from ferroptosis

Intervertebral disc degeneration (IVDD) leads to a series of degenerative spine diseases. Clinical treatment of IVDD is mainly surgery, lacking effective drugs to alleviate intervertebral disc degeneration. In this study, we analysed the mRNA sequencing dataset of human degenerative intervertebral disc tissues and revealed the participation of ferroptosis in IVDD. Furthermore, we confirmed that TNF-α, an important cytokine in IVDD, induces ferroptosis in nucleus pulposus cells. Subsequently, a ferroptosis inhibitors screening strategy using multiple ferroptosis indicators was developed. Through the screen of various natural compounds, cynarin, a natural product enriched in Artichoke, was discovered to inhibit ferroptosis of nucleus pulposus cells. Cynarin can dose-dependently inhibit the catabolism of nucleus pulposus cells, increase the expression of key ferroptosis-inhibiting genes (GPX4 and NRF2), inhibit the increment of cellular Fe2+, lipid peroxides, and reactive oxygen species. It can also prevent mitochondria shrinkage, reduce mitochondria cristae density in ferroptosis, and prevent IVDD in the rat model. In conclusion, cynarin is a potential candidate for the drug development for IVDD.

Inhibiting Heat Shock Protein 90 Attenuates Nucleus Pulposus Fibrosis and Pathologic Angiogenesis Induced by Macrophages via Down-Regulating Cell Migration–Inducing Protein

Intervertebral disc (IVD) degeneration (IVDD) is usually accompanied by nucleus pulposus (NP) fibrosis and pathological angiogenesis, which are possibly associated with macrophage infiltration. Our previous researches revealed the destructive role of macrophages and the protective effects of inhibiting heat shock protein 90 (HSP90) in IVDD. Here, the effects of inhibiting HSP90 on NP fibrosis and pathological angiogenesis induced by macrophages are further investigated. Single-cell RNA sequencing analysis reveals the classification of fibrotic NP cell (NPC) clusters and healthy NPC clusters in human NP tissues, and fibrotic NPC clusters are possibly associated with angiogenesis-related biological processes. Immunostaining shows the spatial association between blood vessel ingrowth and macrophage infiltration, as well as elevated levels of cell migration inducing protein (CEMIP) and vascular endothelial growth factor A (VEGFA) in severely degenerated human IVD tissues. Particularly, HSP90 inhibitor 17-AAG ameliorates macrophage-induced fibrotic phenotype of NPCs via inhibiting CEMIP. M2 but not M1 macrophages promote the angiogenesis ability of endothelial cells (ECs), which is attenuated by 17-AAG or HSP90 siRNA. Reversing the fibrotic phenotype of NPCs by Cemip siRNA also mitigates the pro-angiogenesis effects of M2-CM-treated NPCs. Moreover, the murine IVDD model supports that 17-AAG ameliorates NP fibrosis and EC invasion in IVD tissues. In conclusion, inhibiting HSP90 attenuates two interrelated pathological processes, NP fibrosis and pathological angiogenesis induced by macrophages via downregulating CEMIP.

INTERVERTEBRAL DISC CELLS IN VIVO INTERNALIZE GRAM-POSITIVE BACTERIA: IMPLICATIONS FOR INTERVERTEBRAL DISC DEGENERATION

A key cause of low back pain is the degeneration of the intervertebral disc (IVD). Causality between infection of the IVD and its degenerative process gained great interest over the last decade. Granville Smith et al. (2021) identified 36 articles from 34 research studies investigating bacteria in human IVDs. Bacteria was identified in 27 studies, whereas 9 attributed bacterial presence to contamination. Cutibacterium acnes was the most abundant, followed by coagulase-negative staphylococcus. However, whether bacteria identified were present in vivo or represent perioperative contamination remains unclear. This study investigated whether bacteria are present in IVDs and what potential effects they may have on native disc cells.Immunohistochemical staining for Gram positive bacteria was performed on human IVD tissue to identify presence and characterise bacterial species. Nucleus pulposus (NP) cells in monolayer and 3D alginate were stimulated with LPS and Peptidoglycan (0.1-50 µg/ml) for 48hrs. Following stimulation qPCR for factors associated with disc degeneration including matrix genes, matrix degrading enzymes, cytokines, neurotrophic factors and angiogenic factors and conditioned media collected for ELISA and luminex analysisGram positive bacteria was detected within human IVD tissue. Internalisation of bacteria by NP cells influenced the cell and nuclei morphology. Preliminary results of exposure of NP cells to bacterial components indicate that LPS as well as Peptidoglycan increase IL-8 and ADAMTS-4 gene expression following 48 hours of stimulation with a dose response seen for IL-8 induction by peptidoglycan compared to the control group. Underlining these results, IL-8 protein release was increased for treated groups compared to non-treated control. Further analysis is underway investigating other output measures and additional biological repeats.This study has demonstrated bacteria are present within IVD cells within IVD tissue removed from degenerate IVD and is determining the potential influence of these on disc degeneration.

P8. Responsiveness of patient derived intervertebral disc cells to mechanobiological treatment

P8. Responsiveness of patient derived intervertebral disc cells to mechanobiological treatment

Macrophage polarization regulates intervertebral disc degeneration by modulating cell proliferation, inflammation mediator secretion, and extracellular matrix metabolism.

Macrophage infiltration and polarization have been increasingly observed in intervertebral disc (IVD) degeneration (IDD). However, their biological roles in IDD are still unrevealed. We harvested conditioned media (CM) derived from a spectrum of macrophages induced from THP-1 cells, and examined how they affect nucleus pulposus cells (NPCs) in vitro, by studying cell proliferation, extracellular matrix (ECM) synthesis, and pro-inflammation expression; and in vivo by injection CM in a rat IDD model. Then, high-throughput sequencing was used to detect differentially expressed genes (DEGs). Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) networks were used to further analysis. Higher CCR7+ (M1 marker) and CD206+ (M2 marker) cell counts were found in the degenerated human IVD tissues as compared with the control. Furthermore, the cell co-culture model showed M1CM attenuated NPC proliferation, downregulated the expression of ECM anabolic genes encoding aggrecan and collagen IIα1, upregulated the expression of ECM catabolic genes encoding MMP-13, and inflammation-related genes encoding IL-1β, IL-6, and IL-12, while M2CM showed contrasting trends. In IDD model, higher histological scores and lower disc height index were found following M1CM treatment, while M2CM exhibited opposite results. M1CM injection decreased ECM anabolic and increased ECM catabolic, as well as the upregulation of inflammation-related genes after 8 weeks treatment, while M2CM slowed down these trends. Finally, a total of 637 upregulated and 655 downregulated genes were detected in M1CM treated NPCs, and 975 upregulated genes and 930 downregulated genes in the M2CM groups. The top 30 GO terms were shown and the most significant KEGG pathway was cell cycle in both groups. Based on the PPI analysis, the five most significant hub genes were PLK1, KIF20A, RRM2, CDC20, and UBE2C in the M1CM groups and RRM2, CCNB1, CDC20, PLK1, and UBE2C in the M2CM groups. In conclusion, macrophage polarization exhibited diverse roles in IDD progression, with M1CM exacerbating cell proliferation suppression and IVD degeneration, while M2CM attenuated IDD development. These findings may facilitate the further elucidation of the role of macrophage polarization in IDD, and provide novel insights into the therapeutic potential of macrophages.

Examining the effects of HMG-CoA reductase inhibitors on anabolic and catabolic signaling pathway proteins associated with degenerative disc disease.

Recent evidence suggests that statins, among lipid-lowering drugs, can be used to lower serum cholesterol levels for reducing the risk of cardiovascular disease in individuals with high cholesterol, as well as reducing DNA damage and having anti-ageing and pleiotropic effects. Additionally, nuclear factor kappa B (NF-κB) is reported to be suppressed in statin-administered nucleus pulposus (NP) cells for the prevention of interleukin (IL) -1 beta (IL-1β)-induced apoptosis and extracellular matrix (ECM) degradation. The purpose of this study is the examine whether it is possible for pharmacological synthetic statin agents added into primary cell cultures obtained from human intervertebral disc tissue (IVD) to stop and eliminate tissue degeneration through the anabolic/catabolic signaling pathways associated with inflammation. Pitavastatin and rosuvastatin were added to monolayer grown human primary annulus fibrosus (AF)/NP cells. Cytotoxicity and proliferation analyses were carried out. AF/NP cells and ECM structure were also examined microscopically. In addition, changes in transcription factors and protein expressions of proinflammatory cytokines, which play important roles in anabolic and catabolic pathways associated with inflammation, were analyzed. Decreased proliferation and cell necrosis were observed at the end of 72 hours in the samples, in which statins were added, compared to the samples in the control group to which no pharmacological agent was administered. In addition to this, changes were observed in the expressions of proteins. All results were statistically significant (p<0.05). To better understand the regenerative effects of these two pharmacological agents on degenerated AF/NP cells, there is an urgent need for prospective studies in which different signaling pathways and receptors on these pathways are investigated, apart from IL-1β; NF-κB signaling pathway and SOX9.

Protective Effects of Growth Differentiation Factor-6 on the Intervertebral Disc: An In Vitro and In Vivo Study.

Growth differentiation factors (GDFs) regulate homeostasis by amplifying extracellular matrix anabolism and inhibiting pro-inflammatory cytokine production in the intervertebral disc (IVD). The aim of this study was to elucidate the effects of GDF-6 on human IVD nucleus pulposus (NP) cells using a three-dimensional culturing system in vitro and on rat tail IVD tissues using a puncture model in vivo. In vitro, Western blotting showed decreased GDF-6 expression with age and degeneration severity in surgically collected human IVD tissues (n = 12). Then, in moderately degenerated human IVD NP cells treated with GDF-6 (100 ng/mL), immunofluorescence demonstrated an increased expression of matrix components including aggrecan and type II collagen. Quantitative polymerase chain reaction analysis also presented GDF-6-induced downregulation of pro-inflammatory tumor necrosis factor (TNF)-α (p = 0.014) and interleukin (IL)-6 (p = 0.016) gene expression stimulated by IL-1β (10 ng/mL). Furthermore, in the mitogen-activated protein kinase pathway, Western blotting displayed GDF-6-induced suppression of p38 phosphorylation (p = 0.041) under IL-1β stimulation. In vivo, intradiscal co-administration of GDF-6 and atelocollagen was effective in alleviating rat tail IVD annular puncture-induced radiologic height loss (p = 0.005), histomorphological degeneration (p < 0.001), matrix metabolism (aggrecan, p < 0.001; type II collagen, p = 0.001), and pro-inflammatory cytokine production (TNF-α, p < 0.001; IL-6, p < 0.001). Consequently, GDF-6 could be a therapeutic growth factor for degenerative IVD disease.

Biomarkers in the Degenerative Human Intervertebral Disc Tissue and Blood.

Patients with back pain comprise a large proportion of the outpatient practice among physiatrists. Diagnostic tools are limited to clinical history, physical examinations, and imaging. Nonsurgical treatments are largely empirical, encompassing medications, physical therapy, manual treatments, and interventional spinal procedures. A body of literature is emerging confirming elevated levels of biomarkers including inflammatory cytokines in patients with back pain and/or radiculopathy, largely because the protein assay sensitivity has increased. These biomarkers may serve as tools to assist diagnosis and assess outcomes.The presence of inflammatory mediators in the intervertebral disc tissues and blood helped to confirm the inflammatory underpinnings of back pain related to intervertebral disc degeneration. Literature reviewed here suggests that biomarkers could assist clinical diagnosis and monitor physiological outcomes during and after treatments for spine-related pain. Biomarkers must be measured in a large and diverse asymptomatic population, in the context of age and comorbidities to prevent false-positive tests. These levels can then be rationally compared with those in patients with back disorders including discogenic back pain, radiculopathy, and spinal stenosis. While studies reviewed here used "candidate marker" approaches, future nonbiased approaches in clearly defined patient populations could uncover novel biomarkers in clinical management of patients.

Correlational analysis of chemokine and inflammatory cytokine expression in the intervertebral disc and blood in patients with lumbar disc disease.

The involvement of intervertebral disc (IVD) tissues, whole blood (WB) cytokines, and chemokines in pain in patients with lumbar degenerative disc disease (LDD) is unknown. We investigated the relationships between inflammatory cytokines and chemokines in human IVD tissues and WB samples and their association with pain. Expression levels of chemokines and cytokine gene expression were measured in samples from 20 patients with LDD and compared between IVD tissues and WB samples. The associations between WB chemokine and cytokine gene expression levels and pain intensity (numeric rating scale) were also analyzed. The mRNA of C-C chemokine ligand 20 (CCL20), C-C chemokine receptor 6 (CCR6), interleukin-6 (IL-6), IL-1β, IL-17, and tumor necrosis factor-α (TNF-α) was expressed in degenerated IVD tissues. Pearson's product-moment correlation analysis produced positive correlations between CCR6 and IL-6 expression levels in IVD tissues (r = 0.845, p < 0.001) and WB samples (r = 0.963, p < 0.001). WB IL-6 and CCR6 mRNA expression levels correlated significantly with present pain, maximum pain, and average pain. By contrast, low back pain (LBP) did not correlate with serum chemokine/cytokine expression. This is the first study to report correlations between chemokine and inflammatory cytokine gene expression levels in IVD tissues and WB samples in patients with LDD in relation to pain intensity. WB CCR6 and IL-6 gene expression levels correlated significantly with present pain, maximum pain, and average pain, but not with LBP. These data provide a new understanding of the role of chemokines and inflammatory cytokines in patients with LDD and may lead to new treatment strategies for pain.

M2a Macrophage-Secreted CHI3L1 Promotes Extracellular Matrix Metabolic Imbalances via Activation of IL-13Rα2/MAPK Pathway in Rat Intervertebral Disc Degeneration.

The accumulation of macrophages in degenerated discs is a common phenomenon. However, the roles and mechanisms of M2a macrophages in intervertebral disc degeneration (IDD) have not been illuminated. This study investigated the expression of the M2a macrophage marker (CD206) in human and rat intervertebral disc tissues by immunohistochemistry. To explore the roles of M2a macrophages in IDD, nucleus pulposus (NP) cells were co-cultured with M2a macrophages in vitro. To clarify whether the CHI3L1 protein mediates the effect of M2a macrophages on NP cells, siRNA was used to knock down CHI3L1 transcription. To elucidate the underlying mechanisms, NP cells were incubated with recombinant CHI3L1 proteins, then subjected to western blotting analysis of the IL-13Rα2 receptor and MAPK pathway. CD206-positive cells were detected in degenerated human and rat intervertebral disc tissues. Notably, M2a macrophages promoted the expression of catabolism genes (MMP-3 and MMP-9) and suppressed the expression of anabolism genes (aggrecan and collagen II) in NP cells. These effects were abrogated by CHI3L1 knockdown in M2a macrophages. Exposure to recombinant CHI3L1 promoted an extracellular matrix metabolic imbalance in NP cells via the IL-13Rα2 receptor, along with activation of the ERK and JNK MAPK signaling pathways. This study elucidated the roles of M2a macrophages in IDD and identified potential mechanisms for these effects.

Transcriptional profiling of intervertebral disc in a post-traumatic early degeneration organ culture model.

IntroductionThe goal of this study is to characterize transcriptome changes and gene regulation networks in an organ culture system that mimics early post‐traumatic intervertebral disc (IVD) degeneration.MethodsTo mimic a traumatic insult, bovine caudal IVDs underwent one strike loading. The control group was cultured under physiological loading. At 24 hours after one strike or physiological loading, RNA was extracted from nucleus pulposus (NP) and annulus fibrosus (AF) tissue. High throughput next generation RNA sequencing was performed to identify differentially expressed genes (DEGs) between the one strike loading group and the control group. Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes analyses were performed to analyze DEGs and pathways. Protein‐protein interaction (PPI) network was analyzed with cytoscape software. DEGs were verified using qRT‐PCR. Degenerated human IVD tissue was collected for immunofluorescence staining to verify the expression of DEGs in human disc tissue.ResultsOne strike loading resulted in significant gene expression changes compared with physiological loading. In total 253 DEGs were found in NP tissue and 208 DEGs in AF tissue. Many of the highly dysregulated genes have known functions in disc degeneration and extracellular matrix (ECM) homeostasis. ACTB, ACTG, PFN1, MYL12B in NP tissue and FGF1, SPP1 in AF tissue were verified by qRT‐PCR and immunofluorescence imaging. The identified DEGs were involved in focal adhesion, ECM‐receptor interaction, PI3K‐AKT, and cytokine‐cytokine receptor interaction pathways. Three clusters of PPI networks were identified. GO enrichment revealed that these DEGs were mainly involved in inflammatory response, the ECM and growth factor signaling and protein folding biological process.ConclusionOur study revealed different DEGs, pathways, biological process and PPI networks involved in post‐traumatic IVD degeneration. These findings will advance the understanding of the pathogenesis of IVD degeneration, and help to identify novel biomarkers for the disease diagnosis.

MicroRNA-16 inhibits the lipopolysaccharide-induced inflammatory response in nucleus pulposus cells of the intervertebral disc by targeting TAB3.

IntroductionA variety of inflammatory mediators are produced by the degenerative human intervertebral disc (IVD) tissues spontaneously, suggesting their role in the development of intervertebral disc degeneration (IDD). Our present study was designed to investigate the regulatory effect of microRNA-16 (miR-16) on the lipopolysaccharide (LPS)-induced inflammatory response in nucleus pulposus (NP) cells of the IVD.Material and methodsNP cells were treated with LPS to induce inflammatory responses. The expression of miRNA and genes was determined by qRT-PCR. Western blot and an ELISA kit were used to detect the proteins and protein expression, respectively. A dual luciferase reporter assay was applied to identify the correlation between a miRNA and a gene, and to test nuclear factor-κB (NF-κB) activity.ResultsThe results suggested that miR-16 positively regulated the mRNA and protein expression of extracellular matrix (ECM) genes (including aggrecan and collagen II) in NP cells, while it was negatively correlated with ECM degrading enzymes (including MMP3, MMP13, ADAMTS4, ADAMTS5) and related genes of nitric oxide (NO) reaction. Further studies revealed that miR-16 could oppositely regulate NF-κB and MAPK pathways by directly mediating their upstream gene TAB3.ConclusionsOur study suggested that, in NP cells of the IVD, miR-16 could exert inhibitory effects on the LPS-induced inflammatory response through NF-κB and MAPK pathways by directly mediating TAB3. In this way, miR-16 would play a protective role against LPS-induced IDD and inflammation. Therefore, miR-16 may be a novel therapeutic target for the inhibit of the ECM in the IVD.

Tissue Renin-Angiotensin System (tRAS) Induce Intervertebral Disc Degeneration by Activating Oxidative Stress and Inflammatory Reaction.

Lumbar intervertebral disc degeneration (IDD) has been the major contributor to low back pain (LBP). IDD is an chronic inflammation process, with the activation of plentiful inflammation-related cytokines and ECM degradation-related enzymes. In the past few years, hypertension has been reported to correlate with LBP. In addition, the local tissue renin-angiotensin system (tRAS) has been identified in multiple tissues, including the spinal cord, skin, kidney, heart, and bone. Recently, tRAS has also been established in both bovine and human intervertebral disc tissues, especially in the degenerated disc tissue. However, the exact of tRAS and IDD remains unknown. In this present study, proteomic analysis, molecular biology analysis, and animal model were all used. Firstly, we revealed that tRAS was excessively activated in the human degenerated intervertebral disc tissue via proteomic analysis and molecular biology analysis. Then, in vitro experiment suggested that Ang II could decrease the cell viability of human NP cells and promote NP cell apoptosis, senescence, oxidative stress, and NLRP3 activation in human NP cells. In addition, Ang II could also trigger degeneration and fibrosis phenotype in human NP cells. Finally, the animal model demonstrated that the local activated ACE/Ang II axis in the NP tissue could accelerate IDD in aging spontaneously hypertensive rats (SHR). Collectively, the degenerated intervertebral disc tissue showed excessively activated tRAS, and local activated tRAS could induce NP cell senescence, apoptosis, oxidative stress, and inflammatory reaction to promote IDD. These biological effects of Ang II on human NP cells may provide novel insight into further treatment of IDD.

Investigation of the Potential Effects of Alpha-Lipoic Acid on Human Degenerated Intervertebral Disc Tissue Primary Cell Cultures.

To investigate the supplementation of alpha-lipoic acid (ALA) at the molecular level to determine its effect on primary cell cultures prepared from human intervertebral disc (IVD) tissue in an in vitro environment. Human primary cell cultures were prepared from IVD tissue resected during surgery. While cell cultures without ALA supplementation formed the control group, those with ALA supplementation formed the study group. All cell groups were stained using acridine orange/propidium iodide (AO/PI), and the incidence of apoptotic cell death was determined under a fluorescent microscope. Cell surface morphology and extracellular matrix (ECM) structures were evaluated under an invert light microscope. Simultaneously, cell proliferation was evaluated by MTT?ELISA analysis, and the expressions of chondroadherin (CHAD), cartilage oligomeric protein (COMP), interleukin-1 beta (IL-1?), and matrix metalloproteinase (MMP)-7 and-19, which are genes associated with ECM regulation, were tested using qRT?PCR. The data obtained were evaluated statistically using Tukey?s honestly significant difference (HSD) test after analysis of variance (ANOVA) was performed. The alpha significance value was accepted as < .05. Compared to the cells in the control group, it was observed that both proliferation was suppressed and ECM structures deteriorated in the cells in the study group. Also, it was reported that the all-gene expression levels changed. ALA supplementation can negatively affect human IVD primary cell cultures in an in vitro environment.

The tissue-renin-angiotensin-system of the human intervertebral disc.

Symptomatic intervertebral disc (IVD) degeneration accounts for significant socioeconomic burden. Recently, the expression of the tissue renin-angiotensin system (tRAS) in rat and bovine IVD was demonstrated. The major effector of tRAS is angiotensin II (AngII), which participates in proinflammatory pathways. The present study investigated the expression of tRAS in human IVDs, and the correlation between tRAS, inflammation and IVD degeneration. Human IVD tissue was collected during spine surgery and distributed according to principal diagnosis. Gene expression of tRAS components, proinflammatory and catabolic markers in the IVD tissue was assessed. Hydroxyproline (OHP) and glycosaminoglycan (GAG) content in the IVD tissue were determined. Tissue distribution of tRAS components was investigated by immunohistochemistry. Gene expression of tRAS components such as angiotensin-converting enzyme (ACE), Ang II receptor type 2 (AGTR2), angiotensinogen (AGT) and cathepsin D (CTSD) was confirmed in human IVDs. IVD samples that expressed tRAS components (n = 21) revealed significantly higher expression levels of interleukin 6 (IL-6), tumour necrosis factor α (TNF-α), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 4 and 5 compared to tRAS-negative samples (n = 37). Within tRAS-positive samples, AGT, matrix-metalloproteinases 13 and 3, IL-1, IL-6 and IL-8 were more highly expressed in traumatic compared to degenerated IVDs. Total GAG/DNA content of non-tRAS expressing IVD tissue was significantly higher compared to tRAS positive tissue. Immunohistochemistry confirmed the presence of AngII in the human IVD. The present study identified the existence of tRAS in the human IVD and suggested a correlation between tRAS expression, inflammation and ultimately IVD degeneration.

High-dose TGF-β1 degrades human nucleus pulposus cells via ALK1-Smad1/5/8 activation.

Transforming growth factor β1 (TGF-β1) can promote the proliferation and differentiation of intervertebral disc cells and participates in its repair process. However, whether TGF-β1 engages in the process of disc degeneration has not yet been fully elucidated. The present study aimed to investigate the function of high-dose TGF-β1 on the metabolism of nucleus pulposus cells (NPCs). TGF-β1 levels in human degenerative intervertebral disc tissues and tumor necrosis factor (TNF)-α-induced degenerative NPCs were analyzed. Furthermore, NPCs were treated with TGF-β1 and inhibitors of TGF-β1 receptors [ALK tyrosine kinase receptor (ALK) 1 and ALK5] to determine the effect of the receptors in the mediation of NPC degeneration. The NPC state was determined by the components of secretory collagen I/II, tissue inhibitor of metalloproteinase-3 (TIMP-3) and matrix metalloproteinase (MMP)-13. The mRNA expression of Smad1/2/3/5/8, the downstream gene of TGF-β1 mediated by ALK, was also measured. Results showed that TGF-β1 and ALK1 were positively associated with the degree of degeneration of NP or NPCs in vitro, but negatively associated with ALK5. Furthermore, high-doses of TGF-β1 suppressed collagen II, but enhanced collagen I, TIMP-3, MMP-13, ALK1/5 and Smad1/2/3/5/8 expression. ALK5 inhibition induced the suppression of Smad2/3 and aggravated high-dose TGF-β1-induced NPC degeneration, as shown by the reduction in collagen II and increase in collagen I, TIMP-3 and MMP-13. By contrast, ALK1 inhibition resulted in Smad1/5/8 suppression and alleviated high-dose TGF-β1-induced NPC degeneration. Taken together, it was concluded that high-doses of TGF-β1 contributed to the degeneration of NPCs via the upregulation of ALK1 and Smad1/5/8.

The Role of Cutibacterium acnes in Intervertebral Disc Inflammation.

Recently, the role of infection of the intervertebral disc (IVD) with Cutibacterium acnes (C. acnes) as a contributor to disc-related low back pain (LBP) has been discussed. The aim of this study was to investigate whether and how C. acnes contributes to the inflammatory processes during IVD disease. The prevalence of C. acnes infection in human IVD tissue was determined by aerobic and anaerobic culture. Thereafter, primary human IVD cells were infected with a reference and a clinical C. acnes strain and analyzed for pro-inflammatory markers (gene/protein level). In a subsequent experiment, the involvement of the Toll-like receptor (TLR) pathway was investigated by co-treatment with sparstolonin B, a TLR2/4 inhibitor. We detected C. acnes in 10% of IVD biopsies (with either herniation or degeneration). Stimulating IVD cells with both C. acnes strains strongly and significantly upregulated expression of Interleukin (IL)-1β, IL-6, IL-8, and inducible nitric oxide synthase (iNOS). IL-6, cyclooxygenase (COX)-2, and iNOS expression was reduced upon TLR2/4 inhibition in 3 out of 5 donors, whereby responders and non-responders could not be differentiated by their basal TLR2 or TLR4 expression levels. We demonstrate that exposure of IVD cells to C. acnes induces an inflammatory response that may contribute to the development of discogenic LBP by involving TLR2/4 activation, yet only in a subgroup of patients. Whether the same response will be observed in vivo and where lower inoculums are present remains to be proven in future studies.

Immunohistochemical analysis of protein expression in formalin fixed paraffin embedded human intervertebral disc tissues.

Immunohistochemistry (IHC) is a useful technique for the localization and semiquantification of protein expression within tissues. Adult human intervertebral disc (IVD) tissues contain a large amount of auto‐fluorescence which often makes immunofluorescence techniques inappropriate on tissue samples but can be applied to isolated cell samples. Thus, IHC remains one of, if not the most common application for protein detection within IVD tissue. Immunostaining localizes antigen expression through specific epitope‐antibody interactions. Within the field of IVD research, IHC is commonly used on fresh frozen and paraffin embedded tissues to elucidate the expression of antigens. Here, we discuss the principles of IHC applied to formalin fixed paraffin embedded IVD tissue and supply optimized protocols for antibodies used within our group to guide research within the IVD field.