The intervertebral disc contains intrinsic circadian clocks that are regulated by age and cytokines and linked to 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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Caspase-3 knockout inhibits intervertebral disc degeneration related to injury but accelerates degeneration related to aging

Scientific Reports ◽

10.1038/s41598-019-55709-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Takashi Ohnishi ◽

Katsuhisa Yamada ◽

Koji Iwasaki ◽

Takeru Tsujimoto ◽

Hideaki Higashi ◽

...

Keyword(s):

Intervertebral Disc ◽

Intervertebral Disc Degeneration ◽

Caspase 3 ◽

Treatment Target ◽

Age Related ◽

Different Types ◽

New Treatments ◽

Surgical Treatments ◽

Ivd Degeneration ◽

Primary Effector

AbstractApproximately 40% of people under 30 and over 90% of people 55 or older suffer from moderate-to-severe levels of degenerative intervertebral disc (IVD) disease in their lumbar spines. Surgical treatments are sometimes effective; however, the treatment of back pain related to IVD degeneration is still a challenge; therefore, new treatments are necessary. Apoptosis may be important in IVD degeneration because suppressing cell apoptosis inside the IVD inhibits degeneration. Caspase-3, the primary effector of apoptosis, may be a key treatment target. We analyzed caspase-3’s role in two different types of IVD degeneration using caspase-3 knockout (Casp-3 KO) mice. Casp-3 KO delayed IVD degeneration in the injury-induced model but accelerated it in the age-induced model. Our results suggest that this is due to different pathological mechanisms of these two types of IVD degeneration. Apoptosis was suppressed in the IVD cells of Casp-3 KO mice, but cellular senescence was enhanced. This would explain why the Casp-3 KO was effective against injury-induced, but not age-related, IVD degeneration. Our results suggest that short-term caspase-3 inhibition could be used to treat injury-induced IVD degeneration.

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Mechanisms of Age-Related Decline in Insulin-Like Growth Factor-I Dependent Proteoglycan Synthesis in Rat Intervertebral Disc Cells

Spine ◽

10.1097/00007632-200111150-00005 ◽

2001 ◽

Vol 26 (22) ◽

pp. 2421-2426 ◽

Cited By ~ 64

Author(s):

Shin’ya Okuda ◽

Akira Myoui ◽

Kenta Ariga ◽

Takanobu Nakase ◽

Kazuo Yonenobu ◽

...

Keyword(s):

Growth Factor ◽

Intervertebral Disc ◽

Proteoglycan Synthesis ◽

Insulin Like Growth Factor ◽

Factor I ◽

Age Related ◽

Disc Cells ◽

Growth Factor I

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LRP5-deficiency in OsxCreERT2 mice models intervertebral disc degeneration by aging and compression

10.1101/720656 ◽

2019 ◽

Author(s):

Matthew J. Silva ◽

Nilsson Holguin

Keyword(s):

Transcription Factor ◽

Intervertebral Disc ◽

Wnt Signaling ◽

Cell Fate ◽

Biomechanical Properties ◽

Nucleus Pulposus Cells ◽

Hypertrophic Chondrocyte ◽

Age Related ◽

Ivd Degeneration

ABSTRACTOsterix is a critical transcription factor of mesenchymal stem cell fate, where its loss or loss of WNT signaling diverts differentiation to a chondrocytic lineage. Intervertebral disc (IVD) degeneration activates differentiation of prehypertrophic chondrocyte-like cells and inactivates WNT signaling, but its interacting role with osterix is unclear. First, compared to young-adult (5mo), mechanical compression of old (18mo) IVD induced greater IVD degeneration. Aging (5 vs 12mo) and/or compression reduced the transcription of osterix and notochordal marker T by 40-75%. Compression elevated transcription of hypertrophic chondrocyte marker MMP13 and pre-osterix transcription factor RUNX2, but less so in 12mo IVD. Next, using an Ai9/td reporter and immunohistochemistry, annulus fibrosus and nucleus pulposus cells of 5mo IVD expressed osterix, but aging and compression reduced its expression. Lastly, in vivo LRP5-deficiency in osterix-expressing cells degenerated the IVD, inactivated WNT signaling, reduced the biomechanical properties by 45-70%, and reduced transcription of osterix, notochordal markers and chondrocytic markers by 60-80%. Overall, these data indicate that age-related inactivation of WNT signaling in osterix-expressing cells may limit regeneration by depleting progenitors and attenuating the expansion of chondrocyte-like cells.

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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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The nucleus pulposus microenvironment in the intervertebral disc: the fountain of youth?

10.22203/ecm.v041a46 ◽

2021 ◽

Vol 41 ◽

pp. 707-738

Author(s):

J , Guerrero ◽

◽

S Häckel ◽

AS Croft ◽

S Hoppe ◽

...

Keyword(s):

Tissue Engineering ◽

Intervertebral Disc ◽

Nucleus Pulposus ◽

Treatment Strategies ◽

Disc Disease ◽

Age Related ◽

Key Factor ◽

Fountain Of Youth ◽

And Function ◽

Ivd Degeneration

The intervertebral disc (IVD) is a complex tissue, and its degeneration remains a problem for patients, without significant improvement in treatment strategies. This mostly age-related disease predominantly affects the nucleus pulposus (NP), the central region of the IVD. The NP tissue, and especially its microenvironment, exhibit changes that may be involved at the outset or affect the progression of IVD pathology. The NP tissue microenvironment is unique and can be defined by a variety of specific factors and components characteristic of its physiology and function. NP progenitor cell interactions with their surrounding microenvironment may be a key factor for the regulation of cellular metabolism, phenotype, and stemness. Recently, celltransplantation approaches have been investigated for the treatment of degenerative disc disease, highlighting the need to better understand if and how transplanted cells can give rise to healthy NP tissue. Hence, understanding all the components of the NP microenvironment seems to be critical to better gauge the success and outcomes of approaches for tissue engineering and future clinical applications. Knowledge about the components of the NP microenvironment, how NP progenitor cells interact with them, and how changes in their surroundings can alter their function is summarised. Recent discoveries in NP tissue engineering linked to the microenvironment are also reviewed, meaning how crosstalk within the microenvironment can be adjusted to promote NP regeneration. Associated clinical problems are also considered, connecting bench-to-bedside in the context of IVD degeneration.

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Response of Mesenchymal Stem Cells and Intervertebral Disc Cells to Inflammatory Challenge

ASME 2011 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2011-53937 ◽

2011 ◽

Author(s):

Neena Rajan ◽

Nathaniel Stetson ◽

Passquale Razzano ◽

Mitchell Levine ◽

Daniel Grande ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Intervertebral Disc ◽

Disc Disease ◽

Clinical Model ◽

Injury Model ◽

Tnf Α ◽

Disc Cells ◽

Ivd Degeneration ◽

Pro Inflammatory Cytokines

Human intervertebral disc (IVD) degeneration is accompanied by elevated levels of pro-inflammatory cytokines, particularly IL-1β and TNF-α [1–3]. Cytokine secretion by disc cells increases catabolic breakdown of the tissue, resulting in a positive feedback of disc integrity loss and further inflammation [4–6]. A recent study by our group has shown that severity of degeneration in an injury model can influence the therapeutic effect of cell based repair, such as treatment with mesenchymal stem cells (MSCs) [7]. The goal of this study is to measure the response of MSCs to inflammatory challenge, and to compare this response to that of differentiated disc cells from the nucleus pulposus (NP), annulus fibrosis (AF) and end plate (EP). In this study, we investigated the effects of lipopolysaccharide (LPS) on intervertebral disc cells and MSCs viability, pro-inflammatory cytokine expression and extracellular matrix (ECM) expression. LPS is an endotoxin that induces strong immune responses in animal tissue and hence widely used as a pre-clinical model of inflammation. This approach provides an opportunity to study broad aspects of the physiological inflammatory process observed in degenerative disc disease.

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

BioMed Research International ◽

10.1155/2016/2481712 ◽

2016 ◽

Vol 2016 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Jia-Wen Zhan ◽

Min-Shan Feng ◽

Li-Guo Zhu ◽

Ping Zhang ◽

Jie Yu

Keyword(s):

Gene Expression ◽

Intervertebral Disc ◽

Static Load ◽

Ex Vivo ◽

Compressive Load ◽

Type Ii ◽

Matrix Gene ◽

Motion Segment ◽

Custom Made ◽

Ivd Degeneration

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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Age-dependent microstructural changes of the intervertebral disc: a validation of proteoglycan-sensitive spectral CT

European Radiology ◽

10.1007/s00330-021-08028-z ◽

2021 ◽

Author(s):

Julian Pohlan ◽

Carsten Stelbrink ◽

Matthias Pumberger ◽

Dominik Deppe ◽

Friederike Schömig ◽

...

Keyword(s):

Computed Tomography ◽

Intervertebral Disc ◽

Chondroitin Sulfate ◽

Ex Vivo ◽

Microstructural Analysis ◽

Inverse Correlation ◽

Anulus Fibrosus ◽

Sulfate Content ◽

Age Related ◽

Age Related Changes

Abstract Objective To analyze the two major components of the intervertebral disc (IVD) in an ex vivo phantom, as well as age-related changes in patients. Methods Collagen and chondroitin sulfate were imaged at different concentrations in agar solution. Age-related changes in disc density were retrospectively analyzed in normal-appearing discs in dual-energy computed tomography (DECT) images from a patient cohort with various spinal pathologies (n = 136). All computed tomography (CT) scans were acquired using single-source DECT at 80 and 135 kVp with automatic exposure calculation. In 136 patients, the attenuation of normal-appearing discs on collagen/chondroitin maps (cMaps) correlated with the patients’ age with Pearson’s r using standardized regions of interest in the anterior anulus fibrosus (AAF) and nucleus pulposus (NP). Results DECT collagen mapping revealed concentration-dependent Hounsfield units (HU) of IVD components. For collagen, we found Pearson’s r = 0.9610 (95% CI 0.6789–0.9959), p = 0.0023 at 120 kVe, and r = 0.8824 (95% CI 0.2495–0.9871), p = 0.0199 in cMap. For chondroitin sulfate, Pearson’s r was 0.9583 (95% CI 0.6603–0.9956), p = 0.0026 at 120 kVp, and r = 0.9646 (95% CI 0.7044–0.9963), p = 0.0019 in cMap. Analysis of normal-appearing IVDs revealed an inverse correlation of density with age in the AAF: Pearson’s r = − 0.2294 at 135 kVp (95% CI − 0.4012 to − 0.04203; p=0.0141) and r = − 0.09341 in cMap (95% CI − 0.2777 to 0.09754; p = 0.0003). In the NP, age and density did not correlate significantly at 135 kVp (p = 0.9228) and in cMap (p = 0.3229). Conclusions DECT-based collagen mapping allows microstructural analysis of the two main intervertebral disc components—collagen and chondroitin sulfate. IVD density declines with age, presumably due to a reduction in collagen and chondroitin sulfate content. Age-related alterations of disc microstructure appear most pronounced in the AAF. Key Points • DECT-based collagen mapping allows precise analysis of the two main intervertebral disc components—collagen and chondroitin sulfate. • Intervertebral disc (IVD) density declines with age, presumably due to a reduction in collagen and chondroitin sulfate content. • Age-related alterations of disc microstructure are most pronounced in the anterior anulus fibrosus (AAF).

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Genetic Therapy for Intervertebral Disc Degeneration

International Journal of Molecular Sciences ◽

10.3390/ijms22041579 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1579 ◽

Cited By ~ 1

Author(s):

Eun Roh ◽

Anjani Darai ◽

Jae Kyung ◽

Hyemin Choi ◽

Su Kwon ◽

...

Keyword(s):

Gene Therapy ◽

Animal Models ◽

Intervertebral Disc ◽

Treatment Options ◽

Mammalian Target Of Rapamycin ◽

Chronic Lower Back Pain ◽

Technological Advances ◽

Disc Cells ◽

Ivd Degeneration

Intervertebral disc (IVD) degeneration can cause chronic lower back pain (LBP), leading to disability. Despite significant advances in the treatment of discogenic LBP, the limitations of current treatments have sparked interest in biological approaches, including growth factor and stem cell injection, as new treatment options for patients with chronic LBP due to IVD degeneration (IVDD). Gene therapy represents exciting new possibilities for IVDD treatment, but treatment is still in its infancy. Literature searches were conducted using PubMed and Google Scholar to provide an overview of the principles and current state of gene therapy for IVDD. Gene transfer to degenerated disc cells in vitro and in animal models is reviewed. In addition, this review describes the use of gene silencing by RNA interference (RNAi) and gene editing by the clustered regularly interspaced short palindromic repeats (CRISPR) system, as well as the mammalian target of rapamycin (mTOR) signaling in vitro and in animal models. Significant technological advances in recent years have opened the door to a new generation of intradiscal gene therapy for the treatment of chronic discogenic LBP.

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Effect of TGF-β3 on the Gene Expression of Intervertebral Disc Cells in 3D Pellet Cultures

ASME 2010 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2010-19246 ◽

2010 ◽

Author(s):

C. Houseman ◽

M. Scro ◽

S. Belverud ◽

D. Chen ◽

P. Razzano ◽

...

Keyword(s):

Intervertebral Disc ◽

Tissue Regeneration ◽

Annulus Fibrosus ◽

Culture System ◽

Tissue Repair ◽

Tissue Type ◽

Disc Cells ◽

Ivd Degeneration

Intervertebral disc (IVD) degeneration typically involves changes in the multiple constitutive tissues of the IVD. Many tissue repair efforts have focused on the use of differentiated disc cells or stem cells for the regeneration of an IVD in vitro. Consequently, successful long term culture of human disc cells is pivotal for tissue regeneration of the IVD. The aim of this study is to establish a long-term in vitro culture system for the growth of disc cells that maintain their phenotype based on the anatomical origin (annulus fibrosus (AF), nucleus pulposus (NP), or the vertebral end-plates (EP)). This maintenance of phenotype is crucial for examination of treatment efficacy, which is typically designed to induce regeneration of a single tissue type (i.e. injection of growth factors into the NP or anti-inflammatory treatment of the EPs).

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