Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in an Organ Culture

The development of mechanically active culture systems helps in understanding of the role of mechanical stress in intervertebral disc (IVD) degeneration. Motion segment cultures facilitate the application and control of mechanical loads. The purpose of this study was to establish a culturing method for rabbit IVD motion segments to observe the effect of static load on the whole disc organ. Segments were cultured in custom-made apparatuses under a constant, compressive load (3 kg) for 2 weeks. Tissue integrity, matrix synthesis, and matrix gene expression profile were assessed and compared with fresh one. The results showedex vivoculturing of samples gradually destroyed the morphology. Proteoglycan contents and gene expression were decreased and downregulated obviously. However, immunohistochemical staining intensity and collagen type II gene expression were significantly enhanced and upregulated. In contrast, these trends were reversed under constant compression. These results indicated short-term static load stimulated the synthesis of type II collagen; however, constant compression led to progressive degeneration and specifically to proteoglycan. Through this study a loading and organ-culturing system forex vivorabbit IVD motion segments was developed, which can be used to study the effects of mechanical stimulation on the biology of IVDs and the pathomechanics of IVD degeneration.

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Unravelling the role of Mesenchymal Stem/Stromal Cells Secretome in intervertebral disc degeneration in a pro-inflammatory/degenerative ex vivo model

10.21203/rs.2.22688/v1 ◽

2020 ◽

Author(s):

JR Ferreira ◽

GQ Teixeira ◽

E Neto ◽

C Ribeiro-Machado ◽

AM Silva ◽

...

Keyword(s):

Gene Expression ◽

Intervertebral Disc ◽

Stromal Cells ◽

Ex Vivo ◽

Low Oxygen ◽

Ex Vivo Model ◽

A Cell ◽

New Perspective ◽

Regulated Gene Expression ◽

Ivd Degeneration

Abstract Background: Mesenchymal stem/stromal cells (MSCs) have been increasingly used in clinical trials for intervertebral disc (IVD) degeneration. Here, we aimed to evaluate the potential of a cell-free approach to degenerated IVD, testing if MSCs secretome can stimulate a regenerative response by modulating the IVD inflammatory cascade. Methods: Human bone marrow-derived MSCs were pre-conditioned with IL-1β (10 ng/mL) and low oxygen (6% O2). The secretome of MSCs (MSCsec) was collected after 48h. Bovine IVD tissue explants cultured in pro-inflammatory/degenerative conditions (needle puncture + IL-1β) were treated with MSCsec or co-cultured with MSCs. Results: MSCsec obtained upon IL-1β-pre-conditioning, as well as MSCs co-culture, down-regulated gene expression of pro-inflammatory cytokines, bIL-6 and bIL-8 after 48h, in IVD. IVD matrix degrading enzymes, bMMP1 and bMMP3, were downregulated and upregulated, respectively, in the presence of MSCsec, but not MSCs. After 14 days, MSCsec-treated IVDs revealed increased aggrecan content at the protein level, contrarily to MSCs/IVD co-cultures. Interestingly, IL-1β-preconditioning only, but not IL-1β-IVD, increased gene expression of hADAMTS5 and hTIMP-1in MSCs. Additionally, conditioned medium from MSCsec-treated IVDs did not promote angiogenesis or neurogenesis. In MSCsec-treated IVD, an increase in MCP-3 and GCP-2 was observed, while SDF-1α, TNF-α, IGF-1, Eotaxin 3, FGF-9, MIP-1δ, IFN-γ, IL-5, TNF-β, IL-4, TGF-β1, IL-16, IGFBP-3 and IGFBP-4 were decreased, compared with MSCs/IVD co-cultures. Conclusions: MSCsec obtained upon IL1β-preconditioning, present an immunomodulatory role in degenerated IVD, as well as MSCs. Nevertheless, MSCsec but not MSCs, potentiate aggrecan deposition in IVD in pro-inflammatory/degenerative conditions. This finding can open new perspective on the use of MSCsec as a cell-based/cell-free approach to LBP.

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Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in Ex vivo Organ Culture

Chinese Medical Journal ◽

10.4103/0366-6999.190666 ◽

2016 ◽

Vol 129 (19) ◽

pp. 2338-2346 ◽

Cited By ~ 2

Author(s):

Li-Guo Zhu ◽

Min-Shan Feng ◽

Jia-Wen Zhan ◽

Ping Zhang ◽

Jie Yu

Keyword(s):

Intervertebral Disc ◽

Organ Culture ◽

Nucleus Pulposus ◽

Static Load ◽

Ex Vivo ◽

Motion Segment

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Influence of Angiopoietin Treatment with Hypoxia and Normoxia on Human Intervertebral Disc Progenitor Cell’s Proliferation, Metabolic Activity, and Phenotype

Applied Sciences ◽

10.3390/app11157144 ◽

2021 ◽

Vol 11 (15) ◽

pp. 7144

Author(s):

Muriel C. Bischof ◽

Sonja Häckel ◽

Andrea Oberli ◽

Andreas S. Croft ◽

Katharina A. C. Oswald ◽

...

Keyword(s):

Gene Expression ◽

Intervertebral Disc ◽

Metabolic Activity ◽

Cell Metabolism ◽

Trauma Patients ◽

Low Back ◽

Relative Gene Expression ◽

Progenitor Cell Population ◽

Ivd Degeneration ◽

Relative Gene

Increasing evidence implicates intervertebral disc (IVD) degeneration as a major contributor to low back pain. In addition to a series of pathogenic processes, degenerated IVDs become vascularized in contrast to healthy IVDs. In this context, angiopoietin (Ang) plays a crucial role and is involved in cytokine recruitment, and anabolic and catabolic reactions within the extracellular matrix (ECM). Over the last decade, a progenitor cell population has been described in the nucleus pulposus (NP) of the IVD to be positive for the Tie2 marker (also known as Ang-1 receptor). In this study, we investigated the influence of Ang-1 and Ang-2 on human NP cell (Tie2+, Tie2- or mixed) populations isolated from trauma patients during 7 days in normoxia (21% O2) or hypoxia (≤ 5% O2). At the end of the process, the proliferation and metabolic activity of the NP cells were analyzed. Additionally, the relative gene expression of NP-related markers was evaluated. NP cells showed a higher proliferation depending on the Ang treatment. Moreover, the study revealed higher NP cell metabolism when cultured in hypoxia. Additionally, the relative gene expression followed, with an increase linked to the oxygen level and Ang concentration. Our study comparing different NP cell populations may be the start of new approaches for the treatment of IVD degeneration.

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Developing consistently reproducible intervertebral disc degeneration at rat caudal spine by using needle puncture

Journal of Neurosurgery Spine ◽

10.3171/2009.2.spine08925 ◽

2009 ◽

Vol 10 (6) ◽

pp. 522-530 ◽

Cited By ~ 53

Author(s):

Huina Zhang ◽

Frank La Marca ◽

Scott J. Hollister ◽

Steven A. Goldstein ◽

Chia-Ying Lin

Keyword(s):

Animal Model ◽

Mr Imaging ◽

Intervertebral Disc ◽

Disc Degeneration ◽

Collagen Type ◽

Type Ii ◽

Collagen Type Ii ◽

Anulus Fibrosus ◽

Safranin O ◽

Ivd Degeneration

Object The goal in this study was to develop a convenient, less-invasive animal model to monitor progression of intervertebral disc (IVD) degeneration for future testing of new treatments for disc degeneration. Methods Level 5/6 and 7/8 IVDs of rat caudal spine were stabbed laterally with 18- or 21-gauge hypodermic needles to a depth of 5 mm from the subcutaneous surface with the aid of fluoroscopy. In vivo MR imaging studies were performed at 4, 8, and 12 weeks postsurgery to monitor progression of IVD degeneration. Histological analysis including H & E and safranin O staining, and immunohistochemical studies of collagen type II and bone morphogenetic protein receptor type II (BMPRII) were assessed at 12 weeks postsurgery. Results The 18- and 21-gauge needle–stabbed discs illustrated decreases in both the T2 density and MR imaging index starting at 4 weeks, with no evidence of spontaneous recovery by 12 weeks. Histological staining demonstrated a decreased nucleus pulposus (NP) area, and the NP–anulus fibrosus border became unclear during the progression of disc degeneration. Similar patterns of degenerative signs were also shown in both safranin O– and collagen type II–stained sections. The BMPRII immunohistochemical analysis of stabbed discs demonstrated an increase in BMPRII expression in the remaining NP cells and became stronger in anulus fibrosus with the severity of disc degeneration. Conclusions After introducing an 18- or 21-gauge needle into the NP area of discs in the rat tail, the stabbed disc showed signs of degeneration in terms of MR imaging and histological outcome measurements. Changes in BMPRII expression in this animal model provide an insight for the effectiveness of delivering BMPs into the region responsible for chondrogenesis for disc repair. This convenient, less-invasive, reproducible, and cost-effective model may be a useful choice for testing novel treatments for disc degeneration.

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TonEBP regulates the hyperosmotic expression of aquaporin 1 and 5 in the intervertebral disc

Scientific Reports ◽

10.1038/s41598-021-81838-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

J. W. Snuggs ◽

S. Tessier ◽

R. A. B. Bunning ◽

I. M. Shapiro ◽

M. V. Risbud ◽

...

Keyword(s):

Gene Expression ◽

Intervertebral Disc ◽

Nucleus Pulposus ◽

Central Region ◽

Aquaporin 1 ◽

Enhancer Binding Protein ◽

Ivd Degeneration ◽

Hyperosmotic Environment

AbstractThe central region of the intervertebral disc (IVD) is rich in proteoglycans, leading to a hyperosmotic environment, which fluctuates with daily loading. The cells of the nucleus pulposus (NP cells) have adapted to this environment via the function of tonicity enhancer binding protein (TonEBP), and NP cells have been shown to express several water channels known as aquaporins (AQP). We have previously shown that AQP1 and 5 decrease during IVD degeneration. Here, the regulation of AQP1 and 5 by hyperosmotic conditions and the role of TonEBP in this regulation was investigated. AQP1 and 5 gene expression was upregulated by hyperosmotic conditions mimicking the osmolality of the healthy IVD, which was abrogated by TonEBP knockdown. Furthermore, AQP1 and 5 immunopositivity was significantly reduced in TonEBPΔ/Δ E17.5 mice when compared with wildtype controls, indicating in vivo expression of AQP1 and 5 is controlled at least in part by TonEBP. This hyperosmotic regulation of AQP1 and 5 could help to explain the decreased AQP1 and 5 expression during degeneration, when the osmolality of the NP decreases. Together this data suggests that TonEBP-regulated osmo-adaptation may be disrupted during IVD degeneration when the expression of both AQPs is reduced.

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Influence of Genetic Background and Sex on Gene Expression in the Mouse (Mus musculus) Tail in a Model of Intervertebral Disc Injury

Comparative Medicine ◽

10.30802/aalas-cm-19-000034 ◽

2020 ◽

Vol 70 (2) ◽

pp. 131-139 ◽

Cited By ~ 1

Author(s):

Julie M Brent ◽

Zuozhen Tian ◽

Frances S Shofer ◽

John T Martin ◽

Lutian Yao ◽

...

Keyword(s):

Gene Expression ◽

Intervertebral Disc ◽

Genetic Background ◽

Needle Puncture ◽

Male And Female ◽

Matrix Gene ◽

Disc Injury ◽

Males And Females ◽

Histologic Changes ◽

Compare Gene Expression

To facilitate rational experimental design and fulfill the NIH requirement of including sex as a biologic variable, we examined the influences of genetic background and sex on responses to intervertebral disc (IVD) injury in the mouse tail. The goal of this study was to compare gene expression and histologic changes in response to a tail IVD injury (needle puncture) in male and female mice on the DBA and C57BL/6 (B6) backgrounds. We hypothesized that extracellular matrix gene expression in response to IVD injury differs between mice of different genetic backgrounds and sex. Consistent changes were detected in gene expression and histologic features after IVD injury in mice on both genetic backgrounds and sexes. In particular, expression of col1a1 and adam8 was higher in the injured IVD of DBA mice than B6 mice. Conversely, col2a1 expression was higher in B6 mice than DBA mice. Sex-associated differences were significant only in B6 mice, in which col2a1 expression was greater in male mice than in female. Histologic differences in response to injury were not apparent between DBA and B6 mice or between males and females. In conclusion, mouse tail IVD showed sex- and strain-related changes in gene expression and histology after needle puncture. The magnitude of change in gene expression differed with regard to genetic background and, to a lesser degree, sex.

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The intervertebral disc contains intrinsic circadian clocks that are regulated by age and cytokines and linked to degeneration

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2016-209428 ◽

2016 ◽

Vol 76 (3) ◽

pp. 576-584 ◽

Cited By ~ 51

Author(s):

Michal Dudek ◽

Nan Yang ◽

Jayalath PD Ruckshanthi ◽

Jack Williams ◽

Elzbieta Borysiewicz ◽

...

Keyword(s):

Intervertebral Disc ◽

Clock Gene ◽

Ex Vivo ◽

Conditional Knockout ◽

Circadian Clocks ◽

Diurnal Rhythms ◽

Tissue Specific ◽

Age Related ◽

Disc Cells ◽

Ivd Degeneration

ObjectivesThe circadian clocks are internal timing mechanisms that drive ∼24-hour rhythms in a tissue-specific manner. Many aspects of the physiology of the intervertebral disc (IVD) show clear diurnal rhythms. However, it is unknown whether IVD tissue contains functional circadian clocks and if so, how their dysregulation is implicated in IVD degeneration.MethodsClock gene dynamics in ex vivo IVD explants (from PER2:: luciferase (LUC) reporter mice) and human disc cells (transduced with lentivirus containing Per2::luc reporters) were monitored in real time by bioluminescence photon counting and imaging. Temporal gene expression changes were studied by RNAseq and quantitative reverse transcription (qRT)-PCR. IVD pathology was evaluated by histology in a mouse model with tissue-specific deletion of the core clock gene Bmal1.ResultsHere we show the existence of the circadian rhythm in mouse IVD tissue and human disc cells. This rhythm is dampened with ageing in mice and can be abolished by treatment with interleukin-1β but not tumour necrosis factor α. Time-series RNAseq revealed 607 genes with 24-hour patterns of expression representing several essential pathways in IVD physiology. Mice with conditional knockout of Bmal1 in their disc cells demonstrated age-related degeneration of IVDs.ConclusionsWe have established autonomous circadian clocks in mouse and human IVD cells which respond to age and cytokines, and control key pathways involved in the homeostasis of IVDs. Genetic disruption to the mouse IVD molecular clock predisposes to IVD degeneration. These results support the concept that disruptions to circadian rhythms may be a risk factor for degenerative IVD disease and low back pain.

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

JOR Spine ◽

10.1002/jsp2.1070 ◽

2019 ◽

Vol 2 (4) ◽

Cited By ~ 2

Author(s):

Yulong Wei ◽

Robert J. Tower ◽

Zuozhen Tian ◽

Bhavana Mohanraj ◽

Robert L. Mauck ◽

...

Keyword(s):

Gene Expression ◽

Spatial Distribution ◽

Intervertebral Disc ◽

Type Ii Collagen ◽

Collagen Gene ◽

Type Ii ◽

Collagen Gene Expression

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The Effect of Adenosine on Extracellular Matrix Production in Porcine Intervertebral Disc Cells

Cells Tissues Organs ◽

10.1159/000496186 ◽

2018 ◽

Vol 206 (1-2) ◽

pp. 73-81 ◽

Cited By ~ 2

Author(s):

Xue Yin ◽

Silvia Gonzales ◽

Somya Sha ◽

Howard Levene ◽

Chun-Yuh Huang

Keyword(s):

Gene Expression ◽

Extracellular Matrix ◽

Intervertebral Disc ◽

Adenosine Receptors ◽

Type Ii Collagen ◽

Underlying Mechanism ◽

Receptor Activation ◽

Type Ii ◽

Atp Production ◽

Intracellular Atp

Compressive loading promotes adenosine triphosphate (ATP) production and release by intervertebral disc (IVD) cells. Extracellular ATP can be rapidly hydrolyzed by ectonucleotidases. Adenosine, one of the adenine derivatives of ATP hydrolysis, can modulate diverse cellular actions via adenosine receptors. The objectives of this study were to investigate the effects of exogenous adenosine on the production of extracellular matrix (ECM; i.e., collagen type II and aggrecan) and ATP of IVD cells and explore the underlying mechanism of action. It was found that adenosine treatment significantly upregulated aggrecan and type II collagen gene expression and the ATP level in IVD cells. Dipyridamole, an adenosine transport blocker, completely suppressed the effects of adenosine on the ATP production and ECM gene expression of the IVD cells, whereas antagonists of adenosine receptors did not significantly affect adenosine-treated IVD cells. The findings suggested that elevated intracellular ATP and upregulation of ECM gene expression by adenosine treatment are mainly due to adenosine uptake rather than receptor activation. Since ECM biosynthesis is a high ATP demanding process, supplementing adenosine could be beneficial as IVD cells are able to utilize it to replenish intracellular ATP and sequentially promote ECM production, which is constantly suppressed by limited nutrition supply due to the avascular nature of the IVD.

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Differential regulation of TRP channel gene and protein expression by intervertebral disc degeneration and back pain

Scientific Reports ◽

10.1038/s41598-019-55212-9 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

A. Sadowska ◽

W. Hitzl ◽

A. Karol ◽

P. Jaszczuk ◽

H. Cherif ◽

...

Keyword(s):

Gene Expression ◽

Back Pain ◽

Intervertebral Disc ◽

Protein Expression ◽

Pain Intensity ◽

Trp Channels ◽

Gene Array ◽

Exact Function ◽

Gene And Protein Expression ◽

Ivd Degeneration

AbstractIntervertebral disc (IVD) degeneration and consequent low back pain (LBP) are common and costly pathological processes that require improved treatment strategies. Transient Receptor Potential (TRP) channels constitute a family of multimodal ion channels that have recently emerged as contributors to disc pathologies and were thus proposed as potential therapeutic targets, although limited data on their presence and function in the IVD exist. The purpose of this study was to determine the mRNA and protein expression of TRP channels in non-degenerated and degenerated human IVD tissue (with different pain intensity and chronicity) using gene array, conventional qPCR and immunohistochemistry. We could demonstrate that 26 out of 28 currently known TRP channels are expressed in the IVD on the mRNA level, thereby revealing novel therapeutic candidates from the TRPC, TRPM and TRPML subfamilies. TRPC6, TRPM2 and TRPML1 displayed enhanced gene and protein expression in degenerated IVDs as compared to non-degenerated IVDs. Additionally, the gene expression of TRPC6 and TRPML1 was influenced by the IVD degeneration grade. Pain intensity and/or chronicity influenced the gene and/or protein expression of TRPC6, TRPM2 and TRML1. Interestingly, decreased gene expression of TRPM2 was observed in patients treated with steroids. This study supports the importance of TRP channels in IVD homeostasis and pathology and their possible application as pharmacological targets for the treatment of IVD degeneration and LBP. However, the exact function and activation of the highlighted TRP channels will have to be determined in future studies.

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