Abstract
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.
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.
