The influence of serum, glucose and oxygen on intervertebral disc cell growth in vitro: implications for degenerative disc disease.
BACKGROUND
The avascular nature of the human intervertebral disc (IVD) is thought to play a major role in disc pathophysiology by limiting nutrient supply to resident IVD cells. In the human IVD, the central IVD cells at maturity are normally chondrocytic in phenotype. However, abnormal cell phenotypes have been associated with degenerative disc diseases, including cell proliferation and cluster formation, cell death, stellate morphologies, and cell senescence. Therefore, we have examined the relative influence of possible blood-borne factors on the growth characteristics of IVD cells in vitro.


METHODS
Bovine IVD cells were cultured either in monolayer to encourage cell proliferation or in alginate to induce chondrocytic differentiation. In both culture systems, cells were maintained with or without 20% serum, with or without 320 mg/dL glucose, and in atmospheric levels (~21%) of oxygen or 1% oxygen. Cell proliferation and viability, cell senescence, and collagen immunopositivity were assessed after 7 days. Statistical differences in these growth characteristics were tested using nonparametric analyses (n = 4 samples).


RESULTS
In both culture systems, serum deprivation significantly inhibited IVD cell proliferation and increased cell positivity for senescence-associated beta-galactosidase (SA-beta-gal), a marker of cell senescence. Conversely, IVD cells cultured in the presence of serum, but deprived of glucose, proliferated significantly more rapidly. In alginate cultures, this enhanced cell proliferation (through glucose deprivation) led to the formation of IVD cell clusters. Serum-deprived cells in monolayer, but not in alginate, adopted a stellate appearance. Oxygen deprivation alone had little effect on IVD cell proliferation or survival. Oxygen and glucose deprivation also had no significant effect on SA-beta-gal positivity. IVD cell viability was markedly and significantly decreased in serum-deprived alginate cultures, but in all other conditions remained at or greater than approximately 95%. Glucose deprivation, but not serum or oxygen deprivation, inhibited synthesis of type I and type II collagen, both in monolayer and alginate cultures.


CONCLUSIONS
This study demonstrates that factors present in serum interact with other nutrients, notably glucose, to play a major role in regulating the behaviour of IVD cells. These findings suggest that IVD cell phenotypes seen in degenerative disc disease may arise through the cells' response to altered vascularisation and nutrient supply.
Johnson
William E B
WE
Centre for Spinal Studies, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, Shropshire, SY10 7AG, UK. Eustace.Johnson@rjah.nhs.uk
Stephan
Simon
S
Roberts
Sally
S
D000818:Animals; D002417:Cattle; D049109:Cell Proliferation; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology; D024042:Collagen Type I; D024043:Collagen Type II; D005947:Glucose / Q000378:metabolism*; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D044967:Serum / Q000378:metabolism*; D001616:beta-Galactosidase
D024042:Collagen Type I; D024043:Collagen Type II; D001616:beta-Galactosidase; D005947:Glucose; D010100:Oxygen