Senescent vs. non-senescent cells in the human annulus in vivo: Cell harvest with laser capture microdissection and gene expression studies with microarray analysis

Gruber, Helen E.; Hoelscher, Gretchen L.; Ingram, Jane A.; Zinchenko, Natalia; Hanley, Edward N.

doi:10.1186/1472-6750-10-5

Cited by 33 publications

(27 citation statements)

References 64 publications

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“…(The only assessment of senescence used in this study was SA-β-Gal positivity, which has its limitations as a technique and can fail to differentiate between cells which are senescent from those in crisis (Cristofalo, 2005)). In addition to this, senescence in a cell does not necessarily render the cell non-functional; it remains viable but may have an altered metabolism (Le Maitre et al, 2007;Gruber et al, 2010). This demonstration in vitro that the amount of serum and glucose which is available to disc cells directly infl uences their ability to both proliferate and survive within alginate beads could explain the presence of proliferating (Johnson et al, 2001), senescent (Roberts et al, 2006a), stressed (Sharp et al, 2009) and/or dying cells (Roberts et al, 2006b) in clusters in vivo in human intervertebral discs.…”

Section: Discussionmentioning

confidence: 99%

The influence of nutrient supply and cell density on the growth and survival of intervertebral disc cells in 3D culture

Stephan¹,

Johnson²,

Roberts³

2011

eCM

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The adult human intervertebral disc (IVD) is normally avascular. Changes to the extracellular matrix in degenerative disc disease may promote vascularisation and subsequently alter cell nutrition and disc homeostasis. This study examines the infl uence of cell density and the presence of glucose and serum on the proliferation and survival of IVD cells in 3D culture.Bovine nucleus pulposus (NP) cells were seeded at a range of cell densities (1.25 x10 5 -10 6 cells/mL) and cultured in alginate beads under standard culture conditions (with 3.15 g/L glucose and 10 % serum), or without glucose and/or 20 % serum. Cell proliferation, apoptosis and cell senescence were examined after 8 days in culture.Under standard culture conditions, NP cell proliferation and cluster formation was inversely related to cell seeding density, whilst the number of apoptotic cells and enucleated "ghost" cells was positively correlated to cell seeding density. Increasing serum levels from 10 % to 20 % was associated with increased cluster size and also an increased prevalence of apoptotic cells within clusters. Omitting glucose produced even larger clusters and also more apoptotic and senescent cells.These studies demonstrate that NP cell growth and survival are influenced both by cell density and the availability of serum or nutrients, such as glucose. The observation of clustered, senescent, apoptotic or "ghost" cells in vitro suggests that environmental factors may infl uence the formation of these phenotypes that have been previously reported in vivo. Hence this study has implications for both our understanding of degenerative disc disease and also cell-based therapy using cells cultured in vitro.

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Section: Discussionmentioning

confidence: 99%

The influence of nutrient supply and cell density on the growth and survival of intervertebral disc cells in 3D culture

Stephan¹,

Johnson²,

Roberts³

2011

eCM

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“…Cellular senescence, which is increased in aged and degenerated discs, is regulated by the MAPK pathways (Gruber et al, 2010;Gruber et al, 2007;Roberts et al, 2006). In several cell types, activation of p38 MAPK, for example, is used as a marker of senescence (Chen and Ames, 1994;Iwasa et al, 2003;Papadopoulou and Kletsas, 2011), while constitutive activation of p38 via MKK3 or MKK6 is reported to induce premature senescence via the upregulation of cyclin-dependent kinase inhibitors (Wang et al, 2002;Wu, 2004).…”

Section: K Wuertz Et Al Nf-b and Mapk In The Intervertebral Discsmentioning

confidence: 99%

Inflammatory and catabolic signalling in intervertebral discs: The roles of NF-B and MAP Kinases

Wuertz

Kletsas

et al. 2012

eCM

202

198

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Painful intervertebral disc disease is characterised not only by an imbalance between anabolic (i.e., matrix synthesis) and catabolic (i.e., matrix degradation) processes, but also by infl ammatory mechanisms. The increased expression and synthesis of matrix metalloproteinases and infl ammatory factors is mediated by specifi c signal transduction, in particular the nuclear factor-kappaB (NF-B) and mitogen-activated protein kinase (MAPK)-mediated pathways. NF-B and MAPK have been identifi ed as the master regulators of infl ammation and catabolism in several musculoskeletal disorders (e.g., osteoarthritis), and recently growing evidence supports the importance of these signalling pathways in painful disc disease. With continuing research exploiting in vitro and in vivo model systems to elucidate the roles of these pathways in disc degeneration, it may be possible in the near future to specifi cally target these major infl ammatory / catabolic signalling pathways to treat painful degenerative disc disease. In this perspective, we aim to summarise the current state of knowledge concerning the infl ammatory and catabolic molecular pathways of intervertebral disc disease (IDD), with a detailed description of NF-B and MAP kinase-mediated signal transduction in disc cells. Furthermore, we will discuss the emerging novel molecular treatment modalities for IDD using pharmacological inhibitors targeting these pathways.

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“…An altered gene expression in senescent cells was recently reported [19]; therefore, the expression of transcripts coding for IVD cell phenotypic markers was assessed in degenerate discs in comparison to healthy discs. Currently, there are no specific markers for NP cells, and they have often been considered as chondrocyte-like cells due to ECM similarity.…”

Section: S16mentioning

confidence: 99%

Ex vivo observation of human intervertebral disc tissue and cells isolated from degenerated intervertebral discs

et al. 2012

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Purpose Disc degeneration, and associated low back pain, are a primary cause of disability. Disc degeneration is characterized by dysfunctional cells and loss of proteoglycans: since intervertebral tissue has a limited capacity to regenerate, this process is at present considered irreversible. Recently, cell therapy has been suggested to provide more successful treatment of IVD degeneration. To understand the potential of cells to restore IVD structure/function, tissue samples from degenerated IVD versus healthy discs have been compared. Methods Discal tissue from 27 patients (40.17 ± 11 years) undergoing surgery for degenerative disc disease (DDD), DDD ? herniation and congenital scoliosis, as controls, was investigated. Cells and matrix in the nucleus pulposus (NP) and annulus fibrosus (AF) were characterized by histology.AF-and NP-derived cells were isolated, expanded and characterized for senescence and gene expression. Three-dimensional NP pellets were cultured and stained for glycosaminoglycan formation.Results Phenotypical markers of degeneration, such as cell clusters, chondrons, and collagen disorganization were seen in the degenerate samples. In severe degeneration, granulation tissue and peripheral vascularization were observed. No correlation was found between the Pfirrmann clinical score and the extent of degeneration. Conclusion The tissue disorganization in degenerate discs and the paucity of cells out of cluster/chondron association, make the IVD-derived cells an unreliable option for disc regeneration.

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Senescent vs. non-senescent cells in the human annulus in vivo: Cell harvest with laser capture microdissection and gene expression studies with microarray analysis

Cited by 33 publications

References 64 publications

The influence of nutrient supply and cell density on the growth and survival of intervertebral disc cells in 3D culture

The influence of nutrient supply and cell density on the growth and survival of intervertebral disc cells in 3D culture

Inflammatory and catabolic signalling in intervertebral discs: The roles of NF-B and MAP Kinases

Ex vivo observation of human intervertebral disc tissue and cells isolated from degenerated intervertebral discs

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