Accelerated cellular senescence in degenerate intervertebral discs: a possible role in the pathogenesis of intervertebral disc degeneration

Abstract

Current evidence implicates intervertebral disc degeneration as a major cause of low back pain, although its pathogenesis is poorly understood. Numerous characteristic features of disc degeneration mimic those seen during ageing but appear to occur at an accelerated rate. We hypothesised that this is due to accelerated cellular senescence, which causes fundamental changes in the ability of disc cells to maintain the intervertebral disc (IVD) matrix, thus leading to IVD degeneration. Cells isolated from non-degenerate and degenerate human tissue were assessed for mean telomere length, senescence-associated beta-galactosidase (SA-beta-gal), and replicative potential. Expression of P16INK4A (increased in cellular senescence) was also investigated in IVD tissue by means of immunohistochemistry. RNA from tissue and cultured cells was used for real-time polymerase chain reaction analysis for matrix metalloproteinase-13, ADAMTS 5 (a disintegrin and metalloprotease with thrombospondin motifs 5), and P16INK4A. Mean telomere length decreased with age in cells from non-degenerate tissue and also decreased with progressive stages of degeneration. In non-degenerate discs, there was an age-related increase in cellular expression of P16INK4A. Cells from degenerate discs (even from young patients) exhibited increased expression of P16INK4A, increased SA-beta-gal staining, and a decrease in replicative potential. Importantly, there was a positive correlation between P16INK4A and matrix-degrading enzyme gene expression. Our findings indicate that disc cell senescence occurs in vivo and is accelerated in IVD degeneration. Furthermore, the senescent phenotype is associated with increased catabolism, implicating cellular senescence in the pathogenesis of IVD degeneration.

Figure 1

The expression of senescence biomarkers in vivo. (a) Mean telomere length (MTL) in cells directly extracted from non-degenerate and degenerate human intervertebral discs (IVDs): correlation with age. Samples are from 20 non-degenerate discs (6 aged 37 years, 7 aged 47 years, 2 aged 59 years, 4 aged 62 years, and 1 aged 74 years), 10 intermediate degenerate discs (4 aged 37 years, 1 aged 44 years, 1 aged 49 years, 2 aged 62 years, and 2 aged 74 years), and 1 severely degenerate disc (aged 49 years). Spearman rank correlation P < 0.05. (b) MTL in cells directly extracted from non-degenerate and degenerate human IVDs: effect of degree of degeneration. *Intermediate degenerate samples are significantly different from non-degenerate samples (P < 0.05). Disc samples are as described in (a). Data are shown as average MTL ± standard error of the mean (SEM) for each disease state. (c) Quantification and localisation of p16^INK4a immunopositivity in human IVDs correlated with degree of degeneration. *Samples are significantly different from non-degenerate samples (P < 0.05). Samples are from 11 non-degenerate discs, 6 intermediate degenerate discs, and 5 severely degenerate discs. Averages ± SEM are presented. (d) p16^INK4a immunopositive cells in human IVDs correlated with age. Samples are as detailed in (c). Intermediate degenerate (grades 4 to 7) and severely degenerate (grades 8 to 12) samples are grouped for correlation analysis. Spearman rank correlation for non-degenerate samples P < 0.05 and for degenerate samples P = 0.26. IAF, inner annulus fibrosus; kbp, kilobase pairs; NP, nucleus pulposus; OAF, outer annulus fibrosus.

Figure 2

Senescence biomarker immunohistochemistry. (a) p16^INK4a immunopositivity in the nucleus pulposus of human intervertebral discs. (b) Immunoglobulin G controls were negative. (c) Senescence-associated β-galactosidase staining in directly extracted cells from non-degenerate discs. (d) Senescence-associated β-galactosidase staining in directly extracted cells from degenerate discs (positive cells indicated with arrows). Scale bars = 190 μm (a, b) and 370 μm (c, d).

Figure 3

Senescence biomarkers in human intervertebral disc (IVD) cells in vitro. (a) Cell growth kinetics: cumulative population doublings in nucleus pulposus (NP) cells extracted from non-degenerate and degenerate IVDs. (b) Percentage of life span completed over time in culture of NP cells extracted from non-degenerate and degenerate IVDs. (c) Mean telomere length in NP cells extracted from non-degenerate and degenerate IVDs with increasing population doubling. Samples used consisted of two non-degenerate discs from one post mortem (L2/3: grade 1, L4/5: grade 2; 37-year-old male) and two degenerate discs from one patient undergoing surgery (L4/5: grade 4, L5/S1: grade 8; 49-year-old male).

Figure 4

Correlation of senescent phenotype with expression of matrix-degrading enzymes. (a) Correlation of MMP-13 and p16^INK4a gene expression in human intervertebral disc (IVD) cells. Spearman rank correlation P < 0.05. (b) Correlation of ADAMTS 5 and p16^INK4a gene expression in human IVD cells. Spearman rank correlation P < 0.05. ADAMTS 5, a disintegrin and metalloprotease with thrombospondin motifs 5; MMP-13, matrix metalloproteinase-13.