Abstract
Purpose: To investigate the effect of timed incubations and osmolarity on the cellular protein profile between nucleus pulpous cells and articular chondrocytes to identify possible cellular markers. Both cell types exists in a constantly interchanging environment in which osmolality changes significantly during disc and joint loading.
Methods: Bovine nucleus pulpous and articular chondrocyte cells were isolated and digested with collagenase. The cells were resuspended in alginate beads and incubated in DME medium. DMEM was prepared with increasing osmolarity (280–580mOsm). At T=0,1,3 & 5 cells were collected by dissolving the alginate beads and then washed. Cellular proteins were analysed by large SDS-PAGE, scanned and analysed by computer package. Bands of proteins of interest were cut out for mass spec analyses.
Results: Analysis of whole cells from the nucleus pulpous and articular chondrocytes by SDS-PAGE at T=0 revealed very similar protein patterns. Over 5 days a peak at around 26kDa that appeared in both cell groups. Differences occurred when the cells were incubated with increasing osmolarity. Nucleus pulpous cells showed a loss of peak intensity around 60kDa and 32kDa. Chondrocyte cells showed increased peak intensity around 140kDa, as well two peaks flanking a major peak at around 55kDa.
Conclusion: Incubation of both cell types in alginate beads caused the appearance of a new peak on SDS-PAGE. When both cell types were incubated with increasing osmolarity new protein peaks appeared which may assist in deciphering between the two cell types. Mass spec will identify the protein peaks.
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.
