Effect of nicotine on spinal disc cells: a cellular mechanism for disc degeneration.
METHODS
Experimental investigation to determine the effect of nicotine on intervertebral spinal disc nucleus pulposus (NP) cells cultured in vitro. OBJECTIVES.: To evaluate the effects of nicotine on cell proliferation, extracellular matrix production, and viability of NP cells in three-dimensional alginate constructs cultured in vitro.


BACKGROUND
Numerous studies confirm that smoking is a strong risk factor for back pain. The most widely accepted explanations for the association between smoking and disc degeneration is malnutrition of spinal disc cells by carboxy-hemoglobin-induced anoxia or vascular disease. Nicotine, a constituent of tobacco smoke, present in most body fluids of smokers is known to have detrimental effects on a variety of tissues. It may also be directly responsible for intervertebral disc (IVD) degeneration by causing cell damage in both the nucleus pulposus and anulus fibrosus. The effect of nicotine on IVD cells has not previously been investigated.


METHODS
Bovine chondrocytic intervertebral disc cells were isolated by sequential digestion of nucleus pulposus and seeded in 2% alginate. The constructs were cultured for 21 days either in growth medium containing freebase nicotine (Sigma) at concentrations found in the serum of smokers (25 nmol/L-300 nmol/L) or in standard nicotine free-medium as controls. Samples were collected at time points 3, 7, 14, and 21 days and a quantitative assay was performed for DNA, glycosaminoglycans (GAG), and hydroxyproline. Samples were also processed for qualitative histologic analysis including immunolocalization of collagen types I and II.


RESULTS
There was both a dose- and time-dependent response to nicotine, with constructs cultured in low-nicotine concentration media demonstrating an early increase in DNA, GAG, and collagen content, while constructs cultured in high nicotine concentration media demonstrated a late decrease in these parameters. At 25 nmol/L dose of nicotine, there was a significant increase (P < 0.05) in the above parameters at day 7 compared with the controls. At higher doses, there was a significant dose-dependent decrease (P < 0.05) in these parameters compared to controls; however, this was only significant at day 14 for the 300 nmol/L group and at day 21 for the 100 nmol/L, 200 nmol/L, and 300 nmol/L groups. Adverse morphologic changes were observed on histology, which included reduced cell proliferation, disrupted cell architecture, disintegration of cells, and extracellular matrix. Immunohistochemistry revealed the presence of type I collagen in the extracellular matrix rather than the normal type II collagen seen in the controls.


CONCLUSIONS
Nicotine has an overall detrimental effect on NP disc cells cultured in vitro. There was significant inhibition of cell proliferation and extracellular matrix synthesis. Nicotine in tobacco smoke may have a role in pathogenesis of disc degeneration.
Akmal
Mohammed
M
Institute of Orthopaedics & Royal National Orthopaedic Hospital, Stanmore, UK. M.Akmal@orthopaedics.com
Kesani
Anil
A
Anand
Bobby
B
Singh
Abhinav
A
Wiseman
Mike
M
Goodship
Allen
A
D000464:Alginates; D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D024042:Collagen Type I / Q000096:biosynthesis; D024043:Collagen Type II / Q000096:biosynthesis; D004247:DNA / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D005109:Extracellular Matrix / Q000187:drug effects; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis; D020723:Glucuronic Acid; D006025:Glycosaminoglycans / Q000096:biosynthesis; D006603:Hexuronic Acids; D006909:Hydroxyproline / Q000032:analysis; D007403:Intervertebral Disc / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009538:Nicotine / Q000633:toxicity*; D012907:Smoking / Q000009:adverse effects
D000464:Alginates; D024042:Collagen Type I; D024043:Collagen Type II; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans; D006603:Hexuronic Acids; D009538:Nicotine; D020723:Glucuronic Acid; D004247:DNA; D006909:Hydroxyproline