Effect of oxygen levels on proteoglycan synthesis by intervertebral disc cells.
METHODS
the response of cells from the annulus fibrosus (AF) and nucleus pulposus (NP) to varying oxygen (O2) concentrations was examined when cultured in alginate.


OBJECTIVE
to study the effect of O2 concentration on AF and NP cells.


BACKGROUND
AF and NP cells possess different metabolic profiles in situ. However, it is not clear whether this difference is maintained in in vitro culture conditions. AF and NP cells can respond differently in the different systems, which may differ from the in vivo environment in terms of nutrient supply and O2 levels. In vivo, O2 levels vary from 1% to 5% within the intervertebral disc, and there is evidence that disc cell metabolism can vary with O2 concentrations.


METHODS
an alginate scaffold was seeded with bovine AF or NP cells and maintained in culture for up to 18 days under different O2 concentrations. The sulfated glycosaminoglycan (GAG) content in the culture medium and the expression of aggrecan, type I (COL1A2) and II (COL2A1) collagen genes were analyzed at day 9 and day 18.


RESULTS
in both NP and AF cells cultured either in normoxia (21% O2) or in hypoxia (5% and 1% O2), the GAG content of the culture medium increased with time, though the rate of increase was diminished in 5% O2. With a decrease in O2 levels, the expression of aggrecan mRNA increased in NP cells. There was little effect of O2 on aggrecan mRNA level in AF cells. However, there was a slight decrease with time. Interestingly, aggrecan mRNA levels did not reflect GAG release for either NP or AF cells. There was no effect with time or O2 levels on COL2A1 message in NP cells. The highest Aggrecan/COL2 message ratio for NP cells was with 1% O2, suggesting this to be the best condition for maintaining the NP phenotype. COL1A2 gene expression in NP and AF cells increased with time, but showed little change with O2 levels in NP cells. The highest COL2/COL1 ratio in NP cells was also observed with 1% O2. Finally, NP cells tended to remain localized in the alginate beads, whereas AF cells tended to migrate from the beads.


CONCLUSIONS
both NP and AF cells showed little change in GAG production with O2 levels ranging from 1% to 21%. Disc cell metabolism is not impaired at low O2 concentrations, which appear beneficial to matrix composition. Furthermore, low oxygen may promote a gelatinous NP matrix, whereas increased oxygen levels may promote a fibrous matrix.
Division of Orthopaedic Surgery, McGill University, Lady Davis Institute for Medical Research, Montreal, QC, Canada. fmwale@ldi.jgh.mcgill.ca
Fackson
F
Mwale
Ioana
I
Ciobanu
Demetri
D
Giannitsios
Peter
P
Roughley
Thomas
T
Steffen
John
J
Antoniou
D053674:Aggrecans / Q000235:genetics / Q000378:metabolism; D000464:Alginates; D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques; D015687:Cell Hypoxia; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics / Q000378:metabolism; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D020723:Glucuronic Acid; D006025:Glycosaminoglycans / Q000378:metabolism; D006603:Hexuronic Acids; D007403:Intervertebral Disc / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D010100:Oxygen / Q000494:pharmacology*; D011509:Proteoglycans / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds
D053674:Aggrecans; D000464:Alginates; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D006025:Glycosaminoglycans; D006603:Hexuronic Acids; D011509:Proteoglycans; D012333:RNA, Messenger; D020723:Glucuronic Acid; D003094:Collagen; D010100:Oxygen