The use of SA‐β‐Gal to assess cell senescence in intervertebral disc cells

Abstract

Introduction The intervertebral disc is reported to age faster than other connective tissues with significant degenerative changes already seen in the second decade of life. In other tissues, senescent cells have an altered phenotype, often with a decreased synthetic ability and response to anabolic cytokines. They appear to contribute to age‐related pathologies such as the degeneration of articular cartilage in osteoarthritis. Little is known of cell senescence in the intervertebral disc. In this study, we have investigated the production of senescence‐associated‐ß‐galactosidase (SA‐ß‐Gal), a ‘biomarker’ of cell senescence, in intervertebral disc cells both in cultured populations in vitro, and in vivo, in pathological human discs.Material and methods Intervertebral disc cells were extracted from coccygeal bovine discs and cultured for 3 months. Confluent cell preparations were stained for SA‐β‐Gal (Fenton et al. 2001) at passages 0, 1, 2, 3 and 4. Preparations were fixed in 3% paraformaldehyde in phosphate‐buffered saline (PBS) for 5 min at room temperature and incubated for 24 h at 37 °C in SA‐ß‐Gal solution containing 1 mg/ml 5‐bromo‐4‐chloro‐3‐indolyl ß‐d‐galactopyranoside (X‐Gal, Sigma, Poole, UK), 5 mmol/l potassium ferrocyanide, 5 mmol/l potassium ferricyanide, 150 mmol/l NaCl, 2 mmol/l MgCl2 and 40 mmol/l trisodium citrate, titrated with NaH2PO4 to pH 6.0. Lysosomal (nonsenescent) ß‐galactosidase activity was detected using the same solution but titrated to pH 4.0. After staining, preparations were rinsed in ice‐cold PBS, dehydrated and mounted. Pathological human disc from patients with disc herniations or discogenic back pain were also stained for SA‐β‐Gal (immersing the tissue in the stain solution overnight) then cryosectioning (10 µm thick) and fixing. Articular cartilage was studied for comparison (Price et al. 2002).Results Cultured intervertebral discs demonstrated some staining for SA‐ß‐Gal at all passages investigated, but there was little change in staining with passage number. Cells in most pathological human discs demonstrated staining for SA‐ß‐Gal. Positive cells were seen more commonly in herniated discs [7/9 (78%); mean age: 46 ± 13] than in those removed from patients with discogenic back pain [2/6 (33%); mean age: 32 ± 5].Discussion To our knowledge, this is the first study of cell senescence in intervertebral disc cells. The greatest level was seen in tissue from herniated discs where cell cluster formation and cell proliferation are common (but the mean age of the herniation group was slightly higher than the discogenic back pain group). However, for a tissue demonstrating such significant age‐related degenerative changes, there is surprisingly little expression of SA‐ß‐Gal in comparison with that found in other pathological cartilages. These preliminary data suggest that, unlike the situation in osteoarthritis, early cell senescence is not a major contributing factor in the pathogenesis of disc degeneration.