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.

Purpose: Proteoglycan loss is one of the first signs of disc degeneration. There is increasing interest in developing biological methods for its replacement both by in vivo repair and through tissue engineered constructs. Many factors influence the rate of proteoglycan accumulation. In this study, we examine how physiological levels of extracellular osmolality and oxygen tension influence proteoglycan accumulation in nucleus pulposus cells in a three-dimensional culture system.

Methods: Cells were isolated from the nucleus pulposus of 18–24 month bovine caudal discs. They were cultured for 6 days in alginate beads at 4 million cells/ml in DMEM containing 6% FBS under 0%, 5% and 21% O₂, Medium osmolality was altered by NaCl addition over the range 270–570 mOsm. Cell viability was determined by manual counting using trypan blue. Lactate production was measured enzymatically and glycosaminoglycan (GAG) accumulation was measured using a DMB assay.

Results: There was no difference in the cell viability. Lactate production decreased under hypo- (270 mOsm) after 6 days in culture. After 6 days GAG accumulation was maximal in beads cultured at 5% O₂ in 370 mOsm where GAG accumulation was 86.1% greater than at 21% O₂ and DMEM at standard Osmolarity (270 mOsm).

CONCLUSION: In our model the prevailing osmolality was a powerful regulator of GAG accumulation by cultured nucleus cells. In vivo prevailing osmolality is governed by GAG concentration. These results thus indicate GAG synthesis rates are regulated by GAG concentration, with implications both for the aetiology of degeneration and for tissue engineering.