Abstract
Introduction
Intervertebral disc (IVD) calcification frequently occurs with age as demonstrated by radiographic analysis of elderly spines and is enhanced in patients with scoliosis. In one study by Chanchairujira K. et al (Spine 2004;230:499–503), the degree of disc degeneration as measured by disc space height was significantly correlated with IVD calcification. Several systemic disorders such as hyperparathyroidism and chondrocalcinosis, which are related to calcium handling, arthritis, trauma, vertebral fusion, and infection, have been associated with IVD calcification. Our preliminary data suggest that ionic calcium content and expression of the extracellular calcium-sensing receptor (CaSR) are increased in NP and AF cells of human Thompson graded discs, however, a role in disc degeneration remains unknown.
Material and Methods
Cells: Human NP and AF cells were incubated in culture media containing various concentrations of calcium (1.0, 1.5, 2.5, and 5.0 mM) a CaSR agonist (5 µM), or IL-1β (10 ng/mL) for 7 days. Lysates were extracted and the expression of aggrecan and type II collagen (Col II) were measured by Western blotting. IVDs: Caudal IVDs from the tails of 20- to 24-month-old steers were isolated and the vertebral bone was removed. IVDs were cultured for 4 weeks in culture medium containing calcium (1.0, 2.5, or 5.0 mM), or a CaSR agonist (5 µM). NP and AF tissues were subjected to guanidium extraction and Western blotting was performed to determine the expression of aggrecan and Col II. Histological sections were prepared to determine degree of mineralization by von Kossa staining and expression of alkaline phosphatase.
Results
The expression of aggrecan and Col II decreased dose-dependently in both NP and AF cells following incubation with calcium or the CaSR agonist. A similar phenomenon was observed in the expression of aggrecan and Col II in IVDs following calcium incubation. In addition to decreases in Col II and aggrecan, increase in mineralization and expression of alkaline phosphatase was observed in calcium-incubated IVDs.
Conclusion
Our results suggest that changes in the local concentrations of calcium are not benign, and that activation of CaSR may be a contributing factor in IVD degeneration.