THE EFFECTS OF NICOTINE ON INTERVERTEBRAL DISC CELLS

Abstract

Nicotine is a constituent of tobacco smoke and is present in the body fluids of smokers^1,2. Numerous studies have confirmed that smoking is a strong risk factor for back pain³. The most widely accepted explanation for the association is that smoking leads to malnutrition of spinal discs due to carboxyhaemoglobin formation. However, other constituents of smoke, such as nicotine, may also be responsible for intervertebral disc (IVD) degeneration by leading to cell necrosis in both the nucleus pulposus and annulus fibrosis. Despite evidence suggesting the detrimental effect on a variety of tissues, the effect of nicotine on IVD cells has not previously been investigated. This study investigated the influence of nicotine on the metabolism and viability of IVD cells cultured in vitro.

Bovine nucleus pulposus (NP) intervertebral disc cells were isolated by sequential digestion of caudal spinal disc nuclei with pronase and collagenase and seeded in 2% alginate at 5x10⁶ cells/ml. The constructs were cultured for 21 days in standard culture medium (DMEM + 20% Fetal calf serum) containing free base nicotine (Sigma) at concentrations ranging from 25nM and 300nM, which reflected the normal physiological concentrations found in the serum of smokers. The medium was replaced every 3 days and representative constructs were removed from culture, digested and assayed for DNA, glycosaminoglycan (GAG) and hydroxyproline content at time points 3, 7, 14 and 21 days. Further constructs were processed for standard histology and immunolocalisation of collagen types I, II and chondroitin-6-sulphate.

The results were analysed statistically using an ANOVA test followed by a non-parametric Dunnit’s test. NP cells demonstrated a dose dependent response. At 25nM dose of nicotine there was a significant increase (p< 0.05) in DNA content, GAG and collagen synthesis in the constructs. At 100nM, 200nM and 300nM doses, there was a significant dose dependent decrease (p< 0.05) in all of these parameters compared to controls cultured under nicotine free conditions. In addition, adverse morphological changes were observed on histology, which included reduced cell proliferation, disrupted cell architecture, disintegration of cells and extracellular matrix. Immunohistochemistry showed the production of type I collagen rather than type II collagen as in the controls.

Nicotine has an overall detrimental effect on cultured nucleus pulposus disc cells in vitro. There was significant inhibition of cell proliferation and extracellular matrix synthesis. Nicotine in tobacco smoke may therefore play a role in the aetiology of disc degeneration that leads to back pain in smokers.