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EFFECT OF INTERLEUKIN-1β AND OXYGEN CONCENTRATION ON PROTEOGLYCAN METABOLISM OF NUCLEUS PULPOSUS CELLS IN VITRO



Abstract

Purpose: The inflammatory response around herniated tissue in the epidural space is believed to play a major role in the spontaneous regression of herniated lumbar disc. Numerous macrophages invade the herniated tissue along with newly formed blood vessels which influence oxygen gradient. Inflammatory cytokines such as interleukin-1 are produced by macrophages. These chemical mediators could stimulate disc cells to produce proteases such as MMPs which degrade the intervertebral disc matrix and could hence influence regression of the herniation. Here we have examined the influence of IL-1β and oxygen tension on proteoglycan turnover using a three-dimensional disc-cell culture system.

Methods: Cells were isolated from the nucleus pulposus of 18–24 month bovine caudal discs by enzyme digestion. They were initially cultured for 14 days in alginate beads in DMEM containing 6% FBS at 4.106 cells/ml under 21% oxygen to accumulate matrix. They were then cultured for 6 days under 0% or 21% oxygen and with or without IL-1β. Glycosaminoglycan (GAG) accumulation (as a measure of proteoglycan content) was measured using a DMB assay. Lactate and glucose production were measured using a standard enzymatic method. Rates of sulfated GAG synthesis was measured from rates of 35S-sulfate accumulation. MMP activity was measured using coumarin fluorescent assay.

Results: The results showed that IL-1β had a significant effect on GAG accumulation and production and that its effect was dependent on oxygen tension. GAG production and sulfate incorporation rates decreased in the presence of IL-1β at high oxygen but low oxygen inhibited the effects of this cytokine. MMP activity increased with IL-1β under 21% oxygen, but not at low oxygen.

Conclusion: Exogenous IL-1β can activate MMP activity and digest the extracellular matrix of the disc but only at high oxygen tensions. Angiogenesis as well as inflammation is thus required for resorption of herniations.

Correspondence should be addressed to Ms Alison McGregor, c/o BOA, SBPR at the Royal College of Surgeons, 35–43 Lincoln’s Inn Fields, London WC2A 3PE.