J Korean Soc Spine Surg.  2014 Jun;21(2):84-89. 10.4184/jkss.2014.21.2.84.

Generation of Proinflammatory Mediator of Intervertebral Disc Cells by Nicotine Stimulation

• Seo HY
• Yun JH
• Kim DY

• KMID: 1800403
• DOI: http://doi.org/10.4184/jkss.2014.21.2.84

Abstract

STUDY DESIGN: Experimental investigation in vitro.
OBJECTIVES
To evaluate the relationship between the degeneration of intervertebral disc cells, and low back pain induced by degeneration of intervertebral disc cells and increases in use of proinflammatory mediators via nicotine stimulation. SUMMARY OF LITERATURE REVIEW: Smoking is a leading cause of degeneration of intervertebral disc cells and low back pain. According to the existing literature, nicotine, one of the main ingredients in cigarettes, causes the degeneration of intervertebral disk cells including decrease of glycoprotein through generation of carboxy-hemoglobin, vasoconstriction, and disability of fibrinolysis and changes of metabolism of nucleus pulposus cells.
MATERIALS AND METHODS
Annulus fibrosus of intervertebral disc and knee joint cartilage were collected from pigs; these cells were acquired by gradual enzyme decomposition. Using Trypan blue, concentration and survival rate of cells were examined; cells were inserted on alginate beads for tertiary cultivation. Nicotine was then applied at 0, 50, 100, 200 and 300 nM, respectively, and the samples were cultivated for three, six and nine days, respectively. After collecting culture fluid, it was measured for interleukin(IL)-1beta, IL-6 and IL-8 with the ELISA Test. DNA of cells used for cultivation was quantitated and the amount of the resulting proinflammatory mediator was normalized. The results were then compared with the result of same study on cartilage of porcine knee joints.
RESULTS
For changes of the inflammatory mediator based on the concentration of nicotine, in nicotine stimulation with low concentration of 50 nM and the control group, there was no significant change, while transient increases of inflammatory mediator showed in nicotine stimulation with concentrations of 100, 200 nM, respectively. There was not a significant increase of IL-1beta observed in all nicotine stimulation groups; these were the same results in porcine cartilage study. The level of IL-6 in 200, 300 nM nicotine concentration showed significant increases, respectively. The level of IL-8 in high dose nicotine stimulation groups also showed significant increases of DNA on the sixth day. And in porcine cartilage study group, significant changes were observed in 200, 300 nM, but the absolute value was lower than that of annulus fibrous cells group.
CONCLUSION
Inflammatory mediators such as IL-6 and IL-8 increased as the result of tertiary cultivation of annulus fibrosus cells of porcine intervertebral disk and nicotine stimulation. It is believed that the cells of the disc annulus are more sensitive than articular chondrocytes to nicotine stimulation. This may be the focus of future long-term studies effects of nicotine other inflammatory cytokines.

Keyword

Intervertebral disc; Annulus fibrosus; Proinflammatory mediator; Nicotine

MeSH Terms

Cartilage
Chondrocytes
Cytokines
DNA
Enzyme-Linked Immunosorbent Assay
Fibrinolysis
Glycoproteins
Interleukin-6
Interleukin-8
Intervertebral Disc*
Knee Joint
Low Back Pain
Metabolism
Nicotine*
Smoke
Smoking
Survival Rate
Swine
Tobacco Products
Trypan Blue
Vasoconstriction
Cytokines
DNA
Glycoproteins
Interleukin-6
Interleukin-8
Nicotine
Smoke
Trypan Blue

Figure

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  Fig. 1. DNA contents of alginate bead constructs with porcine annulus fibrosus cells (AF group) and porcine knee joint cartilage cells (AC group), cultured for up to 9 days.

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  Fig. 2. GAG contents of alginate bead constructs with porcine annulus fibrosus cells (AF group) and porcine knee joint cartilage cells (AC group), cultured for up to 9 days.

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  Fig. 3. Measured IL-1β in cultured media with porcine annulus fibrosus cells (AF group) and porcine knee joint cartilage cells (AC group), seeded alginate bead constructs.

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  Fig. 4. Measured IL-6 in cultured media porcine annulus fibrosus cells (AF group) and porcine knee joint cartilage cells (AC group), seeded alginate bead constructs.

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  Fig. 5. Measured IL-8 in cultured media porcine annulus fibrosus cells (AF group) and porcine knee joint cartilage cells (AC group), seeded alginate bead constructs.

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